Methods and Compositions for Treating Bleeding Disorders

ABSTRACT

Aspects of the invention include methods for enhancing blood coagulation in a subject. In practicing methods according to certain embodiments, an amount of a non-anticoagulant sulfated polysaccharide (NASP) is administered to a subject to enhance blood coagulation in the subject. Also provided are methods for preparing a NASP composition having blood coagulation enhancing activity. Compositions and kits for practicing methods of the invention are also described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/111,684 filed May 19, 2011, which is a continuation of U.S.application Ser. No. 13/006,396 filed Feb. 13, 2011; which applicationpursuant to 35 U.S.C. §119 (e), claims priority to U.S. ProvisionalPatent Application Ser. No. 61/335,964, filed Jan. 14, 2010, thedisclosures of which are herein incorporated by reference.

INTRODUCTION

Bleeding is one of the most serious and significant manifestations ofdisease, and may occur from a local site or be systemic. Localizedbleeding may be associated with lesions and may be further complicatedby a defective haemostatic mechanism. Blood clotting is inadequate inbleeding disorders, which may be caused by congenital coagulationdisorders, acquired coagulation disorders, or hemorrhagic conditionsinduced by trauma. Congenital or acquired deficiencies of any of thecoagulation factors may be associated with a hemorrhagic tendency. Somecongenital coagulation disorders include hemophilia, a recessiveX-linked disorder involving a deficiency of coagulation factor VIII(hemophilia A) or factor IX (hemophilia B) and von Willebrands disease,a rare bleeding disorder involving a severe deficiency of vonWillebrands factor. Acquired coagulation disorders may arise inindividuals without a previous history of bleeding as a result of adisease process. For example, acquired coagulation disorders may becaused by inhibitors or autoimmunity against blood coagulation factors,such as factor VIII, von Willebrand factor, factors IX, V, XI, XII andXIII; or by hemostatic disorders such as caused by liver disease, whichmay be associated with decreased synthesis of coagulation factors.

SUMMARY

Aspects of the invention include methods for enhancing blood coagulationin a subject. In practicing methods according to certain embodiments, anamount of a non-anticoagulant sulfated polysaccharide (NASP) isadministered to a subject to enhance blood coagulation in the subject.Also provided are methods for preparing a NASP composition having bloodcoagulation enhancing activity. Compositions and kits for practicingmethods of the invention are also described.

In certain embodiments, the present invention provides a method forenhancing blood coagulation by administering a composition having anamount of a NASP to a subject, where the NASP has a sulfur content of 8%or more by weight. In some instances, the NASP is a fucoidan. Forexample, in these embodiments, the fucoidan may be Fucoidan GFS 5508005,Undaria pinnatifida, depyrogenated; Fucoidan GFS 5508004, Undariapinnatifida; Fucoidan GFS 5508003, Undaria pinnatifida; Fucoidan25307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucusvesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5kD; Fucoidan 5308004, Fucus vesiculosus; Fucoidan 5308005, Fucusvesiculosus; Fucoidan L/FVF1091, Fucus vesiculosus; Fucoidan VG201096A,Fucus vesiculosus; Fucoidan VG201096B, Fucus vesiculosus; Fucoidan VG57,Undaria pinnatifida, high charge (high sulphation, deacetylated);Fucoidan VG50, Ascophyllum nodosum, max. MW peak 149.7 kD; andcombinations thereof.

In some instances, methods of invention further include administering ablood coagulation factor to the subject in conjunction with a NASPhaving a sulfur content of 8% or more. In these instances, the bloodcoagulation factor may include but are not limited to factor Xa, factorIXa, factor XIa, factor XIIa, VIIIa, prekallekrein, and high-molecularweight kininogen, tissue factor, factor VIIa, factor Va, factor Xa,factor II, factor V, factor VII, factor VIII, factor IX, factor X,factor XI, factor XII, factor XIII, von Willebrands factor, andcombinations thereof. For example, in some embodiments, methods of theinvention include administering to a subject an amount of a NASP havinga sulfur content of 8% or more and factor VIII. In another embodiment,methods include administering to a subject an amount of a NASP having asulfur content of 8% or more and factor IX.

In certain embodiments, aspects of the invention also provide methodsfor preparing a NASP having blood coagulation enhancing activity byextracting a NASP from a biological source and increasing the sulfurcontent of the extracted NASP. For example, in some instances, thesulfur content of the NASP may be increased in a manner sufficient toproduce a NASP having a sulfur content of 10% sulfur or more by weight.In other instances, the sulfur content of the NASP may be increased in amanner sufficient to produce a NASP having a sulfur content of 15%sulfur or more by weight.

In certain embodiments, the present invention provides a method forenhancing blood coagulation by administering an amount of a NASP to asubject, where the NASP has 40% or more fucose saccharide residues. Insome instances, the NASP is a fucoidan. For example, in theseembodiments, the fucoidan may be Fucoidan GFS 5508005, Undariapinnatifida, depyrogenated; Fucoidan GFS 5508004, Undaria pinnatifida;Fucoidan VG 23, E. Maxima; Fucoidan L/FVF1093, Fucus vesiculosus,Fucoidan L/FVF1092, Fucus vesiculosus; and combinations thereof.

In certain embodiments, methods of invention further includeadministering a blood coagulation factor to the subject in combinationwith a NASP having 40% or more fucose saccharide residues. In theseinstances, the blood coagulation factor may include but are not limitedto factor Xa, factor IXa, factor XIa, factor XIIa, VIIIa, prekallekrein,and high-molecular weight kininogen, tissue factor, factor VIIa, factorVa, factor Xa, factor II, factor V, factor VII, factor VIII, factor IX,factor X, factor XI, factor XII, factor XIII, von Willebrands factor,and combinations thereof. For example, in one embodiment, methods of theinvention include administering to a subject an amount of a NASP having40% or more fucose saccharide residues and factor VIII. In anotherembodiments, methods include administering to a subject an amount of aNASP having 40% or more fucose saccharide residues and factor IX.

In certain embodiments, the present invention provides a method forenhancing blood coagulation by administering to a subject an amount ofone or more of Fucoidan 5307002, Fucus vesiculosus, max. MW peak 126.7kD; Fucoidan VG49, Fucus vesiculosus, hydrolyzed sample of 5307002 oflower MW, max. MW peak 22.5 kD; Fucoidan VG57, Undaria pinnatifida, highcharge (high sulphation, deacetylated); Fucoidan GFS (5508005), Undariapinnatifida, depyrogenated; Fucoidan GFS (L/FVF-01091), Fucusvesiculosus, depyrogenated, max. MW peak 125 kD; Fucoidan GFS(L/FVF-01092), Fucus vesiculosus, depyrogenated, max. MW peak 260 kD;Fucoidan GFS (L/FVF-01093), Fucus vesiculosus, hydrolyzed depyrogenated,max. MW peak 36 kD; Maritech® Ecklonia radiata extract; Maritech®Ecklonia maxima extract; Maritech® Macrocystis pyrifera extract;Maritech® Immune trial Fucoidan Blend; and combinations thereof.

In certain embodiments, methods of the invention include enhancing bloodcoagulation by administering to a subject an amount of Fucoidan GFS(L/FVF-01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD tothe subject to enhance blood coagulation.

In some instances, methods of invention may further includeadministering a blood coagulation factor to the subject in conjunctionwith one of the fucoidans noted above. In these instances, the bloodcoagulation factor may include but are not limited to factor Xa, factorIXa, factor XIa, factor XIIa, VIIIa, prekallekrein, and high-molecularweight kininogen, tissue factor, factor VIIa, factor Va, factor Xa,factor II, factor V, factor VII, factor VIII, factor IX, factor X,factor XI, factor XII, factor XIII, von Willebrands factor, andcombinations thereof. For example, in some embodiments, methods of theinvention include administering to a subject an amount of Fucoidan GFS(L/FVF-01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD andfactor VIII. In another embodiment, methods include administering to asubject an amount of Fucoidan GFS (L/FVF-01091), Fucus vesiculosus,depyrogenated, max. MW peak 125 kD and factor IX.

In certain embodiments, compositions of the invention decreases bloodclotting time when tested in the dPT assay. In additional embodiments,the compositions of interest display procoagulant activity as determinedusing calibrated automated thrombography (CAT) in Factor VIII and/orFactor IX deficient plasma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mechanism of thrombin generation as as measured usingcalibrated automated thrombography (CAT) in FVIII-inhibited plasma.

FIG. 2 show an example of data acquired for the procoagulant activity offucoidan Fucus vesiculosus L/FVF-1091 as measured using calibratedautomated thrombography (CAT) in FVIII-inhibited plasma.

FIG. 3 shows the experimental setup and mechanism as measured byActivated Partial Thromboplastin Time (aPTT) Assay.

FIG. 4 show an example of data acquired for the pro- and anti-coagulantactivity as measured using the Activated Partial Thromboplastin Time(aPTT) Assay.

FIG. 5 shows an example of data acquisition in determining clottingtime, clot formation time and mean clot formation as measured usingRotation Thromboelastometry.

FIG. 6 shows a second example of data acquisition in determiningclotting time, clot formation time and mean clot formation as measuredusing Rotation Thromboelastometry.

FIG. 7 shows an Ion Chromatogram (IC) for determining monosaccharidecomposition of a fucoidan sample.

FIG. 8 shows monosaccharide composition for several NASPs as measured byIon Chromatography.

FIG. 9 shows monosaccharide composition for several NASPs as measured byIon Chromatography.

FIG. 10 shows monosaccharide composition for several NASPs as measuredby Ion Chromatography.

FIG. 11 shows NMR spectra for determining fucose and alginate contentand heterogeneity of a fucoidan sample.

FIG. 12 shows the experimental setup for CaCo2 bioavailability screeningto determine the % resorption of fucoidans.

FIG. 13 shows an example of the amount of NASP resorbed in CaCo2bioavailability screening for fucoidan Fucus vesiculosus L/FVF-1091.

FIG. 14 shows an example of data acquired for the procoagulant activityof some fucoidans as measured using calibrated automated thrombography(CAT) to determine the mode of TFPI inhibition by fucoidans inFVIII-inhibited plasma.

FIG. 15 shows an example of data acquired for the procoagulant activityof some fucoidans as measured using calibrated automated thrombography(CAT) to determine the mode of TFPI inhibition by fucoidans in normalplasma.

FIG. 16 shows an example of data acquired for the procoagulant activityof some fucoidans as measured using calibrated automated thrombography(CAT) to determine the mode of TFPI inhibition by fucoidans inFVIII-inhibited dFX plasma.

FIG. 17 shows results from studies to probe the interaction of fucoidanwith human TFPI proteins as measured by surface plasmon resonanceexperiments (Biacore 3000, G.E. Healthcare).

RELEVANT DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a “NASP” may include a mixture of two or more NASPs, asdesired.

An “NASP” as used herein refers to sulfated polysaccharide (SP)extracted from a biological source that exhibit non-anticoagulant andanticoagulant activity in any of the various clotting assays describedherein. One measure of activity is to compare the clotting timedemonstrated by a NASP with the anticoagulant activity displayed byheparin. For example, NASPs of interest exhibit anticoagulant activityin the dilute prothrombin time (dPT) or activated partial thromboplastintime (aPTT) clotting assay that is no more than one-third, such as lessthan one-tenth, the molar anticoagulant activity of unfractionatedheparin (MW range 8,000 to 30,000; mean 18,000 daltons). As such, NASPsof interest demonstrate a 2-fold or more lower anticoagulant activity ascompared to heparin, such as a 5-fold or more lower anticoagulantactivity as compared to heparin, such as a 10-fold or more loweranticoagulant activate as compared to heparin, such as a 25-fold or morelower anticoagulant activity as compared to heparin, such as a 50-foldor more lower anticoagulant activity as compared to heparin, including a100-fold or more lower anticoagulant activity as compared to heparin, byemploying methods and compositions as provided herein.

NASPs of interest may range in molecular weight from 10 daltons to1,000,000 daltons, such as for example, from 100 daltons to 900,000daltons, such as from 500 daltons to 500,000 daltons, such as from 1000daltons to 250,000 daltons, including 5000 daltons to 150,000 daltons.Fucoidans may range in average molecular weight from about 10 daltons toabout 500,000 daltons, such as from about 100 daltons to about 300,000daltons, such as from 1000 daltons to 250,000 daltons, including 1000daltons to 150,000 daltons.

NASPs may be used in the methods of the invention for improvinghemostasis, in treating bleeding disorders, such as those associatedwith deficiencies of coagulation factors or for reversing the effects ofanticoagulants. The ability of NASPs to promote clotting and reducebleeding may be determined using various in vitro clotting assays (e.g.,TFPI-dPT, thrombin generation and thromboelastography (TEG) assays) andin vivo bleeding models (e.g. tail snip, transverse cut, whole bloodclotting time, or cuticle bleeding time determination in hemophilic miceor dogs). See, e.g., PDR Staff. Physicians' Desk Reference. 2004,Anderson et al. (1976) Thromb. Res. 9:575-580; Nordfang et al. (1991)Thromb Haemost. 66:464-467; Welsch et al. (1991) Thrombosis Research64:213-222; Broze et al. (2001) Thromb Haemost 85:747-748; Scallan etal. (2003) Blood. 102:2031-2037; Pijnappels et al. (1986) Thromb.Haemost. 55:70-73; and Giles et al. (1982) Blood 60:727-730, and theexamples herein.

A “procoagulant” is used herein in its conventional sense to refer toany factor or reagent capable of initiating or accelerating clotformation. A procoagulant of the invention includes but is not limitedto any activator of the intrinsic or extrinsic coagulation pathways,such as a clotting factor selected from the group consisting of factorXa, factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein,high-molecular weight kininogen, tissue factor, factor VIIa, and factorVa, as well as other reagents that promote clotting include kallikrein,APTT initiator (i.e., a reagent containing a phospholipid and a contactactivator), Russel's viper venom (RVV time), and thromboplastin (fordPT). In some embodiments, contact activators may be employed asprocoagulant reagents. For example, contact activators may includemicronized silica particles, ellagic acid, sulfatides, kaolin or thelike. Procoagulants may be from a crude natural extract, a blood orplasma sample, isolated and substantially purified, synthetic, orrecombinant. Procoagulants may include naturally occurring clottingfactors or fragments, variants or covalently modified derivativesthereof that retain biological activity (i.e., promote clotting).

The term “polysaccharide,” as used herein, refers to a polymercontaining two or more covalently linked saccharide residues. Saccharideresidues may be linked for example by glycosidic, ester, amide, or oximelinking moieties. The average molecular weight of polysaccharides mayvary widely, such as for example ranging from 100 to 1,000,000 daltonsand more, such as 100 to 500,000 daltons and more, such as 1000 to250,000 daltons and more, such as 1000 to 100,000 daltons and more, suchas 10,000 to 50,000 daltons and more. Polysaccharides may be straightchained (i.e., linear) or branched or may contain discrete regions oflinear and branched portions. Polysaccharides may also be fragments ofpolysaccharides generated by degradation (e.g., hydrolysis) of largerpolysaccharides. Degradation can be achieved by any convenient protocolincluding treatment of polysaccharides with acid, base, heat, oxidantsor enzymes to yield fragmented polysaccharides. Polysaccharides may bechemically altered and may be modified, including but not limited to,sulfation, polysulfation, esterification, and methylation.

Molecular weight, as discussed herein, can be expressed as either anumber average molecular weight or a weight average molecular weight.Unless otherwise indicated, all references to molecular weight hereinrefer to the weight average molecular weight. Both molecular weightdeterminations, number average and weight average, can be measured usingfor example, gel permeation chromatography or other liquidchromatography techniques.

The term “derived from” is used herein to identify the original sourceof a molecule but is not meant to limit the method by which the moleculeis made which can be, for example, by chemical synthesis or recombinantmethodologies.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of a reference molecule, that retain desired activity, suchas clotting activity in the treatment of a bleeding disorder. The terms“variant” and “analog” in reference to a polypeptide (e.g., clottingfactor) refer to compounds having a native polypeptide sequence andstructure with one or more amino acid additions, substitutions(generally conservative in nature) and/or deletions, relative to thenative molecule, so long as the modifications do not destroy biologicalactivity and which are “substantially homologous” to the referencemolecule as defined below. The amino acid sequences of such analogs willhave a high degree of sequence homology to the reference sequence, e.g.,amino acid sequence homology of 50% or more, such as 60% or more, suchas 70% or more, such as 80% or more, such as 90% or more, such as 95% ormore, including 99% or more when the two sequences are aligned. In someinstances, analogs will include the same number of amino acids but willinclude substitutions. The term “mutein” further includes polypeptideshaving one or more amino acid-like molecules including but not limitedto compounds contain only amino and/or imino molecules, polypeptidescontaining one or more analogs of an amino acid (including, for example,synthetic non-naturally occurring amino acids, etc.), polypeptides withsubstituted linkages, as well as other modifications known in the art,both naturally occurring and non-naturally occurring (e.g., synthetic),cyclized, branched molecules and the like. The term also includesmolecules comprising one or more N-substituted glycine residues (a“peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S.Pat. Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem.Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA(1992) 89:9367-9371 for descriptions of peptoids). In embodiments of theinvention, analogs and muteins have at least the same clotting activityas the native molecule.

As discussed above, analogs may include substitutions that areconservative, i.e., those substitutions that take place within a familyof amino acids that are related in their side chains. Specifically,amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are in some instances classifiedas aromatic amino acids. For example, an isolated replacement of leucinewith isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar conservative replacement of an amino acidwith a structurally related amino acid, will not have a major effect onthe biological activity. For example, the polypeptide of interest mayinclude up to about 5-10 conservative or non-conservative amino acidsubstitutions, or even up to about 15-25 conservative ornon-conservative amino acid substitutions, or any integer between 5-25,so long as the desired function of the molecule remains intact.

By “derivative” is meant any suitable modification of the referencemolecule of interest or of an analog thereof, such as sulfation,acetylation, glycosylation, phosphorylation, polymer conjugation (suchas with polyethylene glycol), or other addition of foreign moieties, solong as the desired biological activity (e.g., clotting activity,inhibition of TFPI activity) of the reference molecule is retained. Forexample, polysaccharides may be derivatized with one or more organic orinorganic groups. Examples include but are not limited topolysaccharides substituted in at least one hydroxyl group with anothermoiety (e.g., a sulfate, carboxyl, phosphate, amino, nitrile, halo,silyl, amido, acyl, aliphatic, aromatic, or a saccharide group), orwhere a ring oxygen has been replaced by sulfur, nitrogen, a methylenegroup, etc. Polysaccharides may be chemically altered, for example, toimprove procoagulant function. Such modifications may include, but arenot limited to, sulfation, polysulfation, esterification, andmethylation.

By “fragment” is meant a molecule containing a part of the intactfull-length sequence and structure. In some instances, a fragment of apolysaccharide may be generated by degradation (e.g., hydrolysis) of alarger polysaccharide. Active fragments of a polysaccharides of theinvention may include about 2-20 saccharide units of the full-lengthpolysaccharide, such as about 5-10 saccharide units of the full-lengthmolecule, and including any integer between 2 saccharide units and thefull-length molecule, so long as the fragment retains biologicalactivity, such as for example, clotting activity or the ability toinhibit TFPI activity. A fragment of a polypeptide can include aC-terminal deletion, an N-terminal deletion, or an internal deletion ofthe native polypeptide. Active fragments of a particular protein mayinclude, in some embodiments, about 5-10 contiguous amino acid residuesof the full-length molecule or more, such as about 15-25 contiguousamino acid residues of the full-length molecule or more, such as about20-50 contiguous amino acid residues of the full-length molecule ormore, and including any integer between 5 amino acids and thefull-length sequence, so long as the fragment in question retainsbiological activity, such as for example, clotting activity.

By “substantially purified” is meant the isolation of a substance (e.g.,non-anticoagulant sulfated polysaccharide) such that the substanceincludes the majority of the sample in which it resides. For example, asample that is substantially purified contains 50% or more of thesubstance of interest, such as 60% or more of the substance of interest,such as 75% or more of the substance of interest, such as 90% or more ofthe substance of interest, such as 95% or more of the substance ofinterest, including 99% or more of the substance of interest. Anyconvenient protocol may be employed for purifying polysaccharides,polynucleotides, and polypeptides of interest and include, but are notlimited to ultrafiltration, selective precipitation, crystallization,ion-exchange chromatography, affinity chromatography and sedimentationaccording to density.

By “isolated” is meant, when referring to a polysaccharide orpolypeptide, that the indicated molecule is separate and discrete fromthe whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macro-moleculesof the same type.

By “homology” is meant the percent identity between two polypeptidemoieties. As referred to herein, two polypeptide sequences are“substantially homologous” to each other when the sequences exhibitabout 50% or more sequence identity, such as 60% or more sequenceidentity, such as 75% or more sequence identity, such as 85% or moresequence identity, such as 90% or more sequence identity, such as 95% ormore sequence identity, including 99% or more sequence identity. In someembodiments, substantially homologous polypeptides include sequenceshaving complete identity to a specified sequence.

By “identity” is meant an exact subunit to subunit correspondence of twopolymeric sequences. For example, an identical polypeptide is one thathas an exact amino acid-to-amino acid correspondence to anotherpolypeptide or an identical polynucleotide is one that has an exactnucleotide-to-nucleotide correspondence to another polynucleotide.Percent identity can be determined by a direct comparison of thesequence information between two molecules (the reference sequence and asequence with unknown % identity to the reference sequence) by aligningthe sequences, counting the exact number of matches between the twoaligned sequences, dividing by the length of the reference sequence, andmultiplying the result by 100. Any convenient protocol may be employedto determine percent identity between two polymeric sequences, such asfor example, ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence andStructure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedicalResearch Foundation, Washington, D.C., which adapts the local homologyalgorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981for peptide analysis.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are of interest.

The term “patient,” is used in its conventional sense to refer to aliving organism suffering from or prone to a condition that can beprevented or treated by administration of a NASP of the invention, andincludes both humans and non-human animals.

By “biological sample” is meant a sample of tissue or fluid isolatedfrom a subject, including but not limited to, for example, blood,plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid,lymph fluid, samples of the skin, external secretions of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, organs, biopsies and also samples of in vitro cell cultureconstituents including but not limited to conditioned media resultingfrom the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

By “therapeutically effective dose or amount” is meant an amount that,when administered as described herein, brings about the desiredtherapeutic response, such as for example, reduced bleeding or shorterclotting times.

By “bleeding disorder” is meant any disorder associated with excessivebleeding, such as a congenital coagulation disorder, an acquiredcoagulation disorder, administration of an anticoagulant, or a traumainduced hemorrhagic condition. As discussed below, bleeding disordersmay include, but are not limited to, hemophilia A, hemophilia B, vonWillebrand disease, idiopathic thrombocytopenia, a deficiency of one ormore contact factors, such as Factor XI, Factor XII, prekallikrein, andhigh molecular weight kininogen (HMWK), a deficiency of one or morefactors associated with clinically significant bleeding, such as FactorV, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II(hypoprothrombinemia), and von Willebrands factor, a vitamin Kdeficiency, a disorder of fibrinogen, including afibrinogenemia,hypofibrinogenemia, and dysfibrinogenemia, an alpha₂-antiplasmindeficiency, and excessive bleeding such as caused by liver disease,renal disease, thrombocytopenia, platelet dysfunction, hematomas,internal hemorrhage, hemarthroses, surgery, trauma, hypothermia,menstruation, and pregnancy.

DETAILED DESCRIPTION

Aspects of the invention include methods for enhancing blood coagulationin a subject. In practicing methods according to certain embodiments, anamount of a non-anticoagulant sulfated polysaccharide (NASP) isadministered to a subject to enhance blood coagulation in the subject.Also provided are methods for preparing a NASP composition having bloodcoagulation enhancing activity. Compositions and kits for practicingmethods of the invention are also described.

Before the invention is described in greater detail, it is to beunderstood that the invention is not limited to particular embodimentsdescribed herein as such embodiments may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the invention describedherein is not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided might be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featureswhich may be readily separated from or combined with the features of anyof the other several embodiments without departing from the scope orspirit of the invention. Any recited method may be carried out in theorder of events recited or in any other order that is logicallypossible. Although any methods and materials similar or equivalent tothose described herein may also be used in the practice or testing ofthe invention, representative illustrative methods and materials are nowdescribed.

In further describing the subject invention, methods for enhancing bloodcoagulation in a subject are described first in greater detail. Next,methods for preparing a NASP composition having blood coagulationenhancing activity are reviewed. Compositions and kits for practicingmethods of the subject invention are also described.

Methods for Enhancing Blood Coagulation in a Subject

As summarized above, aspects of the invention include methods forenhancing blood coagulation by administering a composition having anamount of a NASP to a subject. The term “enhancing blood coagulation” isused in its conventional sense to refer to accelerating the initiation(i.e., reducing the amount time for coagulation to begin) of bloodcoagulation as well as the overall rate of blood coagulation of thesubject (i.e., reducing the amount of time for blood coagulation to becomplete). In some embodiments, methods of the invention accelerate theinitiation of blood coagulation. For example, methods of the inventionmay reduce the amount of time required for the blood to begincoagulating by 5% or more, such as by 10% or more, such as by 25% ormore, such as by 50% or more, such as by 75% or more, such as by 90% ormore, such as 95% or more, as compared to a suitable control. In otherembodiments, methods of the invention increase the rate of bloodcoagulation. For example, methods of the invention may increase the rateof blood coagulation by 2% or more, such as by 5% or more, such as by10% or more, such as by 25% or more, such as by 50% or more, such as by75% or more, such as by 100% or more, such as by 200% or more, includingby 500% or more, as compared to a suitable control.

In embodiments of the invention, methods for enhancing blood coagulationin a subject are provided. By “subject” is meant the person or organismreceiving the blood coagulation enhancement. As such, subjects of theinvention may include but are not limited to humans and other primates,such as chimpanzees and other apes and monkey species; farm animals suchas cattle, sheep, pigs, goats and horses; domestic mammals such as dogsand cats; laboratory animals including rodents such as mice, rats andguinea pigs; birds, including domestic, wild and game birds such aschickens, turkeys and other gallinaceous birds, ducks, geese, and thelike.

In some embodiments, the subject methods may be employed to treatbleeding disorders, such as a chronic or acute bleeding disorder, acongenital coagulation disorder caused by a blood factor deficiency, anacquired coagulation disorder and administration of an anticoagulant.For example, bleeding disorders may include, but are not limited tohemophilia A, hemophilia B, von Willebrand disease, idiopathicthrombocytopenia, a deficiency of one or more contact factors, such asFactor XI, Factor XII, prekallikrein, and high molecular weightkininogen (HMWK), a deficiency of one or more factors associated withclinically significant bleeding, such as Factor V, Factor VII, FactorVIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia),and von Willebrands factor, a vitamin K deficiency, a disorder offibrinogen, including afibrinogenemia, hypofibrinogenemia, anddysfibrinogenemia, an alpha₂-antiplasmin deficiency, and excessivebleeding such as caused by liver disease, renal disease,thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage,hemarthroses, surgery, trauma, hypothermia, menstruation, and pregnancy.

In other embodiments, the subject methods may be employed to enhanceblood coagulation in order to reverse the effects of an anticoagulant ina subject. For example, the subject may have been treated with ananticoagulant including, but not limited to, heparin, a coumarinderivative, such as warfarin or dicumarol, TFPI, AT III, lupusanticoagulant, nematode anticoagulant peptide (NAPc2), active-siteblocked factor VIIa (factor VIIai), factor IXa inhibitors, factor Xainhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban(DPC906), inhibitors of factors Va and VIIIa, including activatedprotein C (APC) and soluble thrombomodulin, thrombin inhibitors,including hirudin, bivalirudin, argatroban, and ximelagatran. In certainembodiments, the anticoagulant in the subject may be an antibody thatbinds a clotting factor, including but not limited to, an antibody thatbinds to Factor V, Factor VII, Factor VIII, Factor IX, Factor X, FactorXIII, Factor II, Factor XI, Factor XII, von Willebrands factor,prekallikrein, or high molecular weight kininogen (HMWK).

Aspects of the invention include administering to a subject acomposition having an amount of a NASP to enhance blood coagulation. Incertain embodiments, methods of the invention include administering to asubject a composition containing an amount of a NASP having a sulfurcontent that is 8% or more sulfur by weight. For example, the NASP mayhave a sulfur content that is 10% or more sulfur by weight, such as 15%or more sulfur by weight, such as 20% or more sulfur by weight,including 25% or more sulfur by weight. In other embodiments, NASPs ofinterest may contain an amount of sulfur that varies, for exampleranging from 5 to 25% sulfur by weight, such as 5 to 20% sulfur byweight, such as 5 to 15% sulfur by weight, including 10 to 15% sulfur byweight. Any convenient protocol can be employed to determine the sulfurcontent of NASPs of interest. Methods for determining the sulfur contentmay include but is not limited to ion chromatography, gaschromatography, mass spectrometry, inductively coupled plasma, atomicabsorption, inductively coupled plasma mass spectrometry, inductivelycoupled plasma atomic emission spectrometry, flame atomic absorptionspectrometry, graphite furnace atomic absorption spectrometry,acidimetric titration, or any combination thereof.

In embodiments of the invention, the sulfur content of NASPs may bepresent in the form of sulfate. The term “sulfate” is used in itconventional sense refers to the oxyanion of sulfur, SO₄ ²⁻, however,any oxyanion of sulfur having a central sulfur atom bonded to at leastone oxygen atom may be employed, such as sulfite, persulfate,hyposulfate or thiosulfate. The overall amount of sulfate present in theNASP may vary. In certain embodiments, the overall amount of sulfatepresent in NASPs of the invention is 20% or more sulfate by weight, suchas 25% or more sulfate by weight, such as 35% or more sulfate by weight,including 50% or more sulfate by weight. In other embodiments, theoverall amount of sulfate in NASPs ranges, for example from 5 to 50%sulfate by weight, such as 5 to 40% sulfate by weight, such as 5 to 30%sulfate by weight, such as 5 to 25% sulfate by weight, such as 10% to 25sulfate by weight, such as 10 to 20% sulfate by weight, including 10 to15% sulfate by weight. Any convenient protocol can be employed todetermine the amount of sulfation of the NASPs, such as those describedabove for determining sulfur content. For example, methods fordetermining the amount of sulfation may include but is not limited tomass spectrometry, inductively coupled plasma, ion chromatography, gaschromatography, atomic absorption, graphite furnace atomic absorptionspectrometry, inductively coupled plasma mass spectrometry, inductivelycoupled plasma atomic emission spectrometry, flame atomic absorptionspectrometry, acidimetric titration, or any combination thereof.

Each polysaccharide residue of NASPs of interest may have a degree ofsulfation that varies. By “degree of sulfation” is meant the number ofsulfate groups bonded to each saccharide residue on the NASPpolysaccharide backbone. In some embodiments, each polysaccharideresidue (e.g., fucose, galactose, rhamnose, arabinose, glucose, mannose,xylose as described in detail below) may contain one (i.e.,monosulfated) or more (i.e., polysulfated) sulfate moieties. Forexample, in some instances the saccharide residue may be sulfated at the4-position of the saccharide residue. In other instances, the saccharideresidue is sulfated at the 3-position. In certain instances, thesaccharide residue is sulfated at both the 4-position and at the3-position. Each residue may have identical degrees of sulfation (e.g.,all saccharide residues being monosulfated) or may have varying degreesof sulfation (e.g., some saccharide residues having identical sulfationand some saccharide residues having different sulfation). For example,10% or more of the saccharide residues of NASPs of the invention may bemonosulfated, such as 15% or more of the saccharide residues, such as25% or more of the saccharide residues, such as 50% or more of thesaccharide residues, such as 75% or more of the saccharide residues,such as 90% or more of the saccharide residues, such as 95% or more ofthe saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention may be monosulfated. On the otherhand, in some embodiments 10% or more of the saccharide residues ofNASPs of the invention are polysulfated, such as 15% or more of thesaccharide residues, such as 25% or more of the saccharide residues,such as 50% or more of the saccharide residues, such as 75% or more ofthe saccharide residues, such as 90% or more of the saccharide residues,such as 95% or more of the saccharide residues, including 99% or more ofthe saccharide residues of NASPs of the invention may be polysulfated.Where both monosulfated and polysulfated saccharide residues arepresent, the ratio of monosulfated residues to polysulfated residues inNASPs of the invention may vary, and in some instances may range between1:1 and 1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250;1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, themolar ratio of monosulfated residues to polysulfated residues (i.e.,monosulfated saccharide residues:polysulfated saccharide residues) inNASPs of interest may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In someembodiments, the ratio of polysulfated residues to monosulfated residues(i.e., polysulfated saccharide residues:monosulfated saccharideresidues) in the NASPs ranges between 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150;1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, ora range thereof. For example, the ratio of polysulfated saccharideresidues to monosulfated residues in NASPs of interest may range between1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and1:500; or 1:100 and 1:1000. Any convenient protocol can be employed todetermine the sulfation of the NASPs, such as described above. Forexample, methods for determining the degree of sulfation of saccharideresidues may include but is not limited to mass spectrometry, NMRspectroscopy, IR spectroscopy, or any combination thereof.

In some embodiments, saccharide residues of NASPs of interest may besulfated at the 4-position. In other embodiments, the saccharideresidues are sulfated at the 3-position. In certain embodiments, thesaccharide residues are sulfated at the 4-position and at the3-position. For example, 10% or more of the saccharide residues of NASPsof the invention may be sulfated at the 4-position, such as 15% or moreof the saccharide residues, such as 25% or more of the saccharideresidues, such as 50% or more of the saccharide residues, such as 75% ormore of the saccharide residues, such as 90% or more of the saccharideresidues, such as 95% or more of the saccharide residues, including 99%or more of the saccharide residues of NASPs of the invention may besulfated at the 4-position. In other embodiments 10% or more of thesaccharide residues of NASPs of the invention are sulfated at the3-position, such as 15% or more of the saccharide residues, such as 25%or more of the saccharide residues, such as 50% or more of thesaccharide residues, such as 75% or more of the saccharide residues,such as 90% or more of the saccharide residues, such as 95% or more ofthe saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention are sulfated at the 3-position. Incertain embodiments 10% or more of the saccharide residues of NASPs ofthe invention are sulfated at both the 3-position and the 4-position,such as 15% or more of the saccharide residues, such as 25% or more ofthe saccharide residues, such as 50% or more of the saccharide residues,such as 75% or more of the saccharide residues, such as 90% or more ofthe saccharide residues, such as 95% or more of the saccharide residues,including 99% or more of the saccharide residues of NASPs of theinvention are sulfated at both the 3-position and the 4-position. Whereboth saccharide residues sulfated at the 4-position and saccharideresidues sulfated at the 3-position are present, the ratio of saccharideresidues sulfated at the 4-position to saccharide residues sulfated atthe 3-position may vary, and in some instances may range between 1:1 and1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the molarratio of saccharide residues sulfated at the 4-position to saccharideresidues sulfated at the 3-position in NASPs of interest may rangebetween 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50and 1:500; or 1:100 and 1:1000. In some embodiments, the ratio ofsaccharide residues sulfated at the 3-position to saccharide residuessulfated at the 4-position in the NASPs ranges between 1:1 and 1:2.5;1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the ratio ofsaccharide residues sulfated at the 3-position to saccharide residuessulfated at the 4-position in NASPs of interest may range between 1:1and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500;or 1:100 and 1:1000. Any convenient protocol can be employed todetermine the type of sulfated saccharide residues of the NASPs, such asdescribed above. For example, methods for determining the degree ofsulfation of saccharide residues may include but is not limited to massspectrometry, NMR spectroscopy, IR spectroscopy, or any combinationthereof.

In certain embodiments, NASPs of the invention may be extracted from abiological source. By “biological source” is meant a naturally-occurringorganism or part of an organism. For example, NASPs of interest may beextracted from plants, animals, fungi or bacteria. In particular, NASPsof interest may be extracted from edible seaweeds, brown algae,echinoderms (e.g., sea urchins, sea cucumbers) and the like. Anyconvenient protocol can be employed for extracting the NASP from thebiological source. For instance, the NASP can be extracted from thebiological source by acid-base extraction, enzymatic degradation,selective precipitation, filtration, among other procedures. Methods forextracting and isolating NASPs from biological sources such as edibleseaweeds and brown algae are described in detail in co-pending U.S.patent application Ser. No. 12/449,712, filed Feb. 25, 2010, thedisclosure of which is herein incorporated by reference, in itsentirety.

In some instances NASPs extracted from a biological source are fucoidanshaving a sulfur content of 8% sulfur or more by weight. For example,fucoidans of interest may include but are not limited to Fucoidan GFS5508005, Undaria pinnatifida, depyrogenated; Fucoidan GFS 5508004,Undaria pinnatifida; Fucoidan GFS 5508003, Undaria pinnatifida; Fucoidan5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucusvesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5kD; Fucoidan 5308004, Fucus vesiculosus; Fucoidan 5308005, Fucusvesiculosus; Fucoidan L/FVF1091, Fucus vesiculosus; Fucoidan VG201096A,Fucus vesiculosus; Fucoidan VG201096B, Fucus vesiculosus; Fucoidan VG57,Undaria pinnatifida, high charge (high sulphation, deacetylated);Fucoidan VG50, Ascophyllum nodosum, max. MW peak 149.7 kD; and anycombinations thereof.

In certain embodiments, aspects of the invention include enhancing bloodcoagulation in a subject by administering to the subject, a compositionthat contains an amount of a NASP having a sulfur content that is 8% ormore sulfur by weight in combination with a blood coagulation factor.For example, the subject may be administered an amount of a compositioncontaining a NASP having a sulfur content that is 8% or more sulfur byweight and one or more blood coagulation factors which include, but arenot limited to factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, factor VIIa, and von Willebrandsfactor, factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa,prekallekrein, and high-molecular weight kininogen, tissue factor,factor VIIa, factor Va, and factor Xa.

Where a composition that contains a NASP having a sulfur content that is8% sulfur or more by weight and a blood coagulation factor isadministered to the subject, the mass ratio of the composition thatcontains a NASP having a sulfur content that is 8% sulfur or more byweight to the blood coagulation factor ranges between 1:1 and 1:2.5;1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratioof the composition that contains a NASP having a sulfur content that is8% sulfur or more by weight to the blood coagulation factor may rangebetween 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio ofthe blood coagulation factor to the composition that contains a NASPhaving a sulfur content that is 8% sulfur or more by weight rangesbetween 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. Forexample, the mass ratio of the blood coagulation factor to thecomposition that contains a NASP having a sulfur content that is 8%sulfur or more by weight may range between 1:1 and 1:10; 1:5 and 1:25;1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

The blood coagulation factor and the composition that contains a NASPhaving a sulfur content that is 8% sulfur or more by weight may beadministered to the subject in any order. In some instances, thecomposition that contains a NASP having a sulfur content that is 8%sulfur or more by weight is administered prior to administering theblood coagulation factor. In other instances, the composition thatcontains a NASP having a sulfur content that is 8% sulfur or more byweight is administered in conjunction with administering the bloodcoagulation factor. In yet other instances, the composition thatcontains a NASP having a sulfur content that is 8% sulfur or more byweight is administered after administering the blood coagulation factor.Where the composition that contains a NASP having a sulfur content thatis 8% sulfur or more by weight is administered in conjunction with theblood coagulation factor, the composition that contains a NASP having asulfur content that is 8% sulfur or more by weight may be mixed with theblood coagulation factor before administering the composition to thesubject. Any convenient mixing protocol may be used, such as a by dryshaking, solution or suspension mixing, industrial mixing protocols andthe like.

In some embodiments, methods of the invention also include extracting aNASP from a biological source and increasing the sulfur content of theextracted NASP. As described above, any convenient protocol can beemployed for extracting the NASP from the biological source. Forexample, the NASP can be extracted from the biological source byacid-base extraction, enzymatic degradation, selective precipitation,filtration, among other procedures. Methods for extracting and isolatingNASPs from biological sources such as edible seaweeds and brown algae isdescribed in detail in co-pending U.S. patent application Ser. No.12/449,712, filed Feb. 25, 2010, the disclosure of which is hereinincorporated by reference, in its entirety.

In some embodiments, the NASP extracted from the biological source mayhave a natural sulfur content that is 8% or more sulfur by weight. Forexample, the NASP extracted from the biological source may have anatural sulfur content that is 10% or more sulfur by weight, such as 15%or more sulfur by weight, such as 20% or more sulfur by weight,including 25% or more sulfur by weight. In other embodiments, the NASPextracted from the biological source may have a sulfur content that isless than 8% sulfur by weight, such as less than 5% sulfur by weight,such as less than 2% sulfur by weight, such as less than 1% sulfur byweight, including less than 0.5% sulfur by weight.

In certain embodiments, the NASP extracted from the biological source ischemically sulfated in a manner sufficient to obtain a NASP having asulfur content of 8% sulfur or more by weight. For example the NASPextracted from the biological source may be chemically sulfated in amanner to obtain a NASP having 10% sulfur or more by weight, such as 15%sulfur or more by weight, such as 20% sulfur or more by weight,including 25% sulfur or more by weight. As such, methods of theinvention increase the sulfur content of the NASP extracted from thebiological source. For example, the sulfur content of NASPs extractedfrom a biological source may be increased by 0.5% sulfur by weight ormore, such as 1% sulfur by weight or more, such as 2% or more sulfur byweight, such as 5% or more sulfur by weight, such as 10% or more sulfurby weight, such as 15% or more sulfur by weight, such as 20% or moresulfur by weight, including 25% or more sulfur by weight. In theseembodiments, the resulting NASPs may have 1.5-fold more sulfur by weightthan the NASP extracted from the biological source, such as 2-fold moresulfur by weight, such as 5-fold more sulfur by weight, such as 10-foldmore sulfur by weight, such as 25-fold more sulfur by weight, including100-fold more sulfur by weight that the NASP extracted from thebiological source.

Any convenient protocol can be used to chemically sulfate the NASPextracted from the biological source, so long as the sulfur content ofthe resulting NASP is 8% sulfur or more by weight and the increasedsulfur content is the result of new sulfate moieties covalently bondedto the NASP structure. In these embodiments, any free hydroxyl grouplocated on the saccharide backbone of the extracted NASP can be modifiedby sulfation to produce a mono- or poly- (e.g., di-substituted) sulfatedsaccharide. For example, one or more free hydroxyl groups along thesaccharide backbone may be sulfated by bonding one or more sulfateanions to the free hydroxyl groups along the saccharide backbone. Inother instances, sulfur trioxide complexes with pyridine, triethylamine,or with stannous complexes may be employed (see for example, methods forsulfating hydroxyl groups in Calvo-Asin, J. A., et al., J. Chem. Soc,Perkin Trans 1, 1997, 1079).

As discussed above, aspects of the invention include administering to asubject a composition having an amount of a NASP to enhance bloodcoagulation. In certain embodiments, methods of the invention includeadministering to a subject a composition having an amount of a NASP thatcontains 40% or more fucose saccharide residues. The saccharide contentof NASPs of interest may vary. In some instances, the saccharide contentof NASPs of interest may include, but is not limited to fucose residues,xylose residues, galactose residues, glucose residues, mannose residues,rhamnose residues, arabinose residues and uronic acid. In someembodiments, NASPs of interest are composed of two or more of fucoseresidues, xylose residues, galactose residues, glucose residues, mannoseresidues, rhamnose residues, arabinose residues and uronic acid. Theamount of each saccharide residue in NASPs of interest may vary. Forexample, 40% or more of the saccharide residues of NASPs of theinvention may be fucose saccharide residues, such as 45% or more of thesaccharide residues, such as 50% or more of the saccharide residues,such as 65% or more of the saccharide residues, such as 75% or more ofthe saccharide residues, such as 90% or more of the saccharide residues,such as 95% or more of the saccharide residues, including 99% or more ofthe saccharide residues of NASPs of the invention may be fucosesaccharide residues. In other instances, 1% or more of the saccharideresidues of NASPs of the invention may be galactose saccharide residues,such as 5% or more of the saccharide residues, such as 10% or more ofthe saccharide residues, such as 15% or more of the saccharide residues,such as 20% or more of the saccharide residues, including 25% or more ofthe saccharide residues of NASPs of the invention may be galactosesaccharide residues. In yet other instances, 1% or more of thesaccharide residues of NASPs of the invention may be uronic acidsaccharide residues, such as 5% or more of the saccharide residues, suchas 10% or more of the saccharide residues, such as 15% or more of thesaccharide residues, such as 20% or more of the saccharide residues,including 25% or more of the saccharide residues of NASPs of theinvention may be uronic acid saccharide residues. Any convenientprotocol can be employed to determine the saccharide content of NASPs ofinterest. Methods for determining the saccharide content may include butis not limited to ion chromatography, gas chromatography, massspectrometry, nuclear magnetic resonance spectroscopy, or anycombination thereof.

In embodiments of the invention, NASPs of interest may be a linear(i.e., unbranched) polysaccharide or may be a branched polysaccharide.In certain instances, NASPs may have portions of its structure that islinear and other parts of its structure that is branched. By “linearpolysaccharide” is meant a polysaccharide or part of a polysaccharidethat contains only α-1,4 glycosidic bonds, or α-1,3 glycosidic bonds, oralternating α-1,3/α-1,4 glycosidic bonds. By “branched polysaccharide”is meant a polysaccharide or part of a polysaccharide that contains twoor more glycosidic bonds to other saccharide residues, where one of theglycosidic bonds is an α-1,4-glycosidic bond or α-1,3 glycosidic bonds,or alternating α-1,3/α-1,4 glycosidic bonds, and the other is anα-1,6-glycosidic bond. The amount of branching in NASPs of interest mayvary.

Aspects of the invention include enhancing blood coagulation in asubject by administering to the subject, a composition having an amountof a NASP that contains 40% or more fucose saccharide residues. In theseembodiments, NASPs of interest may contain 45% or more fucose saccharideresidues, such as 50% or more fucose saccharide residues, such as 60% ormore fucose saccharide residues, such as 75% or more fucose saccharideresidues, such as 85% or more fucose saccharide residues, such as 90% ormore fucose saccharide residues, including 95% or more fucose saccharideresidues. In other embodiments, NASPs administered to the subject maycontain an amount of fucose saccharides residues that ranges, forexample from 40 to 99% fucose saccharide residues, such as 40 to 90%fucose saccharide residues, such as 45 to 85% fucose saccharideresidues, such as 50 to 80% fucose saccharide residues, such as 50% to75% fucose saccharide residues, including 50 to 60% fucose saccharideresidues.

In certain embodiments, NASPs of interest may contain 40% or moresulfated esters of fucose saccharide residues, such as 50% or moresulfated esters of fucose saccharide residues, such as 60% or moresulfated esters of fucose saccharide residues, such as 75% or moresulfated esters of fucose saccharide residues, such as 85% or moresulfated esters of fucose saccharide residues, such as 90% or moresulfated esters of fucose saccharide residues, including 95% or moresulfated esters of fucose saccharide residues. As described in detailabove, sulfated esters of fuose saccharide residues may vary in theamount of sulfation, regioselectivity of sulfation as well as degree ofsulfation. For example, sulfated esters of fucose saccharide residuesmay, in some instances, be monosulfated. In other instances, sulfatedesters of fucose saccharide residues may be polysulfated. Likewise, incertain instances, sulfated esters of fucose saccharide residues may besulfated that the 4-position. On the other hand, sulfated esters offucose saccharide residues may be sulfated at the 3-position.

In certain embodiments, NASPs of interest contain 40% or more fucosesaccharide residues and 20% or more galactose saccharide residues, suchas 45% or more fucose saccharide residues and 20% or more galactoseresidues, such as 50% or more fucose saccharide residues and 20% or moregalactose residues, such as 60% or more fucose saccharide residues and20% or more galactose residues, such as 70% or more fucose saccharideresidues and 20% or more galactose residues. In other embodiments, NASPsof interest contain 40% or more fucose saccharide residues and 25% ormore galactose saccharide residues, such as 40% or more fucosesaccharide residues and 30% or more galactose saccharide residues, andincluding 40% or more fucose saccharide residues and 40% or moregalactose saccharides residues.

As described above, NASPs of the invention may be extracted from abiological source. In some instances NASPs extracted from a biologicalsource may be fucoidans that contain 40% or more fucose saccharideresidues. In certain embodiments, fucoidans of interest may include butare not limited to Fucoidan GFS 5508005, Undaria pinnatifida,depyrogenated; Fucoidan GFS 5508004, Undaria pinnatifida; Fucoidan VG23, E. Maxima; Fucoidan L/FVF1093, Fucus vesiculosus, FucoidanL/FVF1092, Fucus vesiculosus; and any combinations thereof.

In certain embodiments, aspects of the invention include enhancing bloodcoagulation in a subject by administering to the subject, a compositionhaving an amount of a NASP that contains 40% or more fucose saccharideresidues in combination with a blood coagulation factor. For example,the subject may be administered an amount of a composition containing aNASP that contains 40% or more fucose saccharide residues and one ormore blood coagulation factors which include, but are not limited tofactor XI, factor XII, prekallikrein, high molecular weight kininogen(HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factorXIII, factor II, factor VIIa, and von Willebrands factor, factor Xa,factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein, andhigh-molecular weight kininogen, tissue factor, factor VIIa, factor Va,and factor Xa.

Where a composition having a NASP that contains 40% or more fucosesaccharide residues and a blood coagulation factor are administered tothe subject, the mass ratio of the composition having a NASP thatcontains 40% or more fucose saccharide residues to blood coagulationfactor may vary, and in some instances may range between 1:1 and 1:2.5;1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratioof the composition having a NASP that contains 40% or more fucosesaccharide residues to blood coagulation factor may range between 1:1and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500;or 1:100 and 1:1000. In some embodiments, the mass ratio of the bloodcoagulation factor to the composition having a NASP that contains 40% ormore fucose saccharide residues ranges between 1:1 and 1:2.5; 1:2.5 and1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and1:1000, or a range thereof. For example, the mass ratio of the bloodcoagulation factor to the composition having a NASP that contains 40% ormore fucose saccharide residues may range between 1:1 and 1:10; 1:5 and1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and1:1000.

The composition having a NASP that contains 40% or more fucosesaccharide residues and the blood coagulation factor may be administeredto the subject in any order. In some instances, the composition having aNASP that contains 40% or more fucose saccharide residues isadministered prior to administering the blood coagulation factor (i.e.,sequentially, on the same day, on different days, etc.). In otherinstances, the composition having a NASP that contains 40% or morefucose saccharide residues is administered in conjunction withadministering the blood coagulation factor. In yet other instances, thecomposition having a NASP that contains 40% or more fucose saccharideresidues is administered after administering the blood coagulationfactor (i.e., sequentially, on the same day, on different days, etc.).Where the composition having a NASP that contains 40% or more fucosesaccharide residues is administered in conjunction with the bloodcoagulation factor, the composition having a NASP that contains 40% ormore fucose saccharide residues may be mixed with the blood coagulationfactor before administering the composition to the subject. Anyconvenient mixing protocol may be used, such as a by dry shaking,solution or suspension mixing, industrial mixing protocols and the like.

Aspects of the invention also include a method of enhancing bloodcoagulation in a subject by administering a composition having an amountof a fucoidan, where the fucoidan is extracted from a biological source.In certain embodiments, methods of the invention include administering acomposition having an amount of a fucoidan selected from the groupconsisting of the compounds from Table 1. In these embodiments,fucoidans of interest may include, but are not limited to Fucoidan5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucusvesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5kD; Fucoidan VG57, Undaria pinnatifida, high charge (high sulphation,deacetylated); Fucoidan GFS (5508005), Undaria pinnatifida,depyrogenated; Fucoidan GFS (L/FVF-01091), Fucus vesiculosus,depyrogenated, max. MW peak 125 kD; Fucoidan GFS (L/FVF-01092), Fucusvesiculosus, depyrogenated, max. MW peak 260 kD; Fucoidan GFS(L/FVF-01093), Fucus vesiculosus, hydrolyzed depyrogenated, max. MW peak36 kD; Maritech® Ecklonia radiata extract; Maritech® Ecklonia maximaextract, Maritech® Macrocystis pyrifera extract; Maritech® Immune trialFucoidan Blend; and combinations thereof.

In these embodiments, aspects of the invention may also includeadministering the composition having an amount of a fucoidan incombination with a blood coagulation factor. For example, the subjectmay be administered an amount of a composition containing a fucoidan andone or more blood coagulation factors which include, but are not limitedto factor XI, factor XII, prekallikrein, high molecular weight kininogen(HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factorXIII, factor II, factor VIIa, and von Willebrands factor, factor Xa,factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein, andhigh-molecular weight kininogen, tissue factor, factor VIIa, factor Va,and factor Xa.

Where a composition having an amount of a fucoidan and blood coagulationfactor are both administered to the subject, the mass ratio of thecomposition having an amount of a fucoidan to blood coagulation factormay vary, and in some instances may range between 1:1 and 1:2.5; 1:2.5and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100;1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500;1:500 and 1:1000, or a range thereof. For example, the mass ratio of thecomposition having an amount of a fucoidan to blood coagulation factormay range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, themass ratio of the blood coagulation factor to the composition having anamount of a fucoidan ranges between 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150;1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, ora range thereof. For example, the mass ratio of the blood coagulationfactor to the composition having an amount of a fucoidan may rangebetween 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50and 1:500; or 1:100 and 1:1000.

The composition having an amount of a fucoidan and the blood coagulationfactor may be administered to the subject in any order. In someinstances, the composition having an amount of a fucoidan isadministered prior to administering the blood coagulation factor (i.e.,sequentially, on the same day, on different days, etc.). In otherinstances, the composition having an amount of a fucoidan isadministered in conjunction with administering the blood coagulationfactor. In yet other instances, the composition having an amount of afucoidan is administered after administering the blood coagulationfactor (i.e., sequentially, on the same day, on different days, etc.).Where the composition having an amount of a fucoidan is administered inconjunction with the blood coagulation factor, the composition having anamount of a fucoidan may be mixed with the blood coagulation factorbefore administering the composition to the subject. Any convenientmixing protocol may be used, such as a by dry shaking, solution orsuspension mixing, industrial mixing protocols and the like.

In certain embodiments of the invention, methods and compositions fortreating bleeding disorders using NASPs as procoagulants are provided.NASPs as disclosed herein can be administered alone (i.e., as singleagents), or in combination with one another, or with other hemostaticagents. As desired, NASPs of interest may be employed in the treatmentof a subject that has been diagnosed as having a bleeding disorder,including congenital coagulation disorders, acquired coagulationdisorders, administration of an anticoagulant, and trauma inducedhemorrhagic conditions.

In some instances, a subject may be diagnosed as having a blood clottingdisorders that includes, but is not limited to hemophilia A, hemophiliaB, von Willebrand disease, idiopathic thrombocytopenia, a deficiency ofone or more contact factors, such as Factor XI, Factor XII,prekallikrein, and high molecular weight kininogen (HMWK), a deficiencyof one or more factors associated with clinically significant bleeding,such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, FactorXIII, Factor II (hypoprothrombinemia), and von Willebrands factor, avitamin K deficiency, a disorder of fibrinogen, includingafibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, analpha₂-antiplasmin deficiency, and excessive bleeding such as caused byliver disease, renal disease, thrombocytopenia, platelet dysfunction,hematomas, internal hemorrhage, hemarthroses, surgery, trauma,hypothermia, menstruation, and pregnancy.

In other instances, a subject may be diagnosed as having a bloodclotting disorder that includes a congenital coagulation disorder or anacquired coagulation disorder caused by a blood factor deficiency. Forexample, the blood factor deficiency may be caused by deficiencies ofone or more factors, including but not limited to, factor V, factor VII,factor VIII, factor IX, factor XI, factor XII, factor XIII, and vonWillebrand factor.

In yet other instances, a subject may be diagnosed as having a bloodclotting disorder resulting from the administration of an anticoagulantto the subject. For example, the anticoagulant may include but is notlimited to, heparin, a coumarin derivative, such as warfarin ordicumarol, tissue factor pathway inhibitor (TFPI), antithrombin III,lupus anticoagulant, nematode anticoagulant peptide (NAPc2), active-siteblocked factor VIIa (factor VIIai), factor IXa inhibitors, factor Xainhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban(DPC906), inhibitors of factors Va and VIIIa, including activatedprotein C (APC) and soluble thrombomodulin, thrombin inhibitors,including hirudin, bivalirudin, argatroban, and ximelagatran. In certainembodiments, the anticoagulant may be an antibody that binds a clottingfactor, including but not limited to, an antibody that binds to FactorV, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II,Factor XI, Factor XII, von Willebrands factor, prekallikrein, or highmolecular weight kininogen (HMWK).

In practicing methods of the invention, protocols for enhancing bloodcoagulation in a subject may vary, such as for example by age, weight,severity of the blood clotting disorder, the general health of thesubject, as well as the particular composition and concentration of theNASPs being administered.

In embodiments of the invention, the concentration of NASPs achieved ina subject following administration may vary, in some instances, rangingfrom 0.01 nM to 500 nM. NASPs of interest are procoagulant at theiroptimal concentration. By “optimal concentration” is meant theconcentration in which NASPs exhibit the highest amount of procoagulantactivity. Since many of the NASPs also demonstrated anticoagulantactivity at much higher concentrations than the optimal concentration,NASPs of the invention show non-anticoagulant behavior in the range ofits optimal concentration. As such, depending on the potency of the NASPas well as the desired effect, the optimal concentration of NASPsprovided by methods of the invention may range, from 0.01 nM to 500 nM,such as 0.1 nM to 250 nM, such as 0.1 nM to 100 nM, such as 0.1 nM to 75nM, such as 0.1 nM to 50 nM, such as 0.1 nM to 25 nM, such as 0.1 nM to10 nM, and including 0.1 nM to 1 nM. Optimal concentrations and activitylevel as determined by calibrated automated thrombography (CAT) assay ofNASPs of interest are summarized in Tables 2-4 below.

Therefore, the dosage of compositions containing NASPs of interest mayvary, ranging from about 0.01 mg/kg to 500 mg/kg per day, such as from0.01 mg/kg to 400 mg/kg per day, such as 0.01 mg/kg to 200 mg/kg perday, such as 0.1 mg/kg to 100 mg/kg per day, such as 0.01 mg/kg to 10mg/kg per day, such as 0.01 mg/kg to 2 mg/kg per day, including 0.02mg/kg to 2 mg/kg per day. In other embodiments, the dosage may rangefrom 0.01 to 100 mg/kg four times per day (QID), such as 0.01 to 50mg/kg QID, such as 0.01 mg/kg to 10 mg/kg QID, such as 0.01 mg/kg to 2mg/kg QID, such as 0.01 to 0.2 mg/kg QID. In other embodiments, thedosage may range from 0.01 mg/kg to 50 mg/kg three times per day (TID),such as 0.01 mg/kg to 10 mg/kg TID, such as 0.01 mg/kg to 2 mg/kg TID,and including as 0.01 mg/kg to 0.2 mg/kg TID. In yet other embodiments,the dosage may range from 0.01 mg/kg to 100 mg/kg two times per day(BID), such as 0.01 mg/kg to 10 mg/kg BID, such as 0.01 mg/kg to 2 mg/kgBID, including 0.01 mg/kg to 0.2 mg/kg BID. The amount of compoundadministered will depend on the potency and concentration of thespecific NASP, the magnitude or procoagulant effect desired, as well asthe route of administration.

As discussed above, compositions containing a NASP as provided bymethods of the invention may be administered in combination with otherNASPs or other therapeutic agents, such as hemostatic agents, bloodfactors, or other medications according to a dosing schedule relying onthe judgment of the clinician and needs of the subject. As such, dosingschedules may include, but is not limited to administration five timesper day, four times per day, three times per day, twice per day, onceper day, three times per week, twice per week, once per week, twice permonth, once per month, and any combination thereof.

In some embodiments, the bleeding disorder may be a chronic condition(e.g., a congenital or acquired coagulation factor deficiency) requiringthe subject methods and compositions in multiple doses over an extendedperiod. Alternatively, methods and compositions of the invention may beadministered to treat an acute condition (e.g., bleeding caused bysurgery or trauma, or factor inhibitor/autoimmune episodes in subjectsreceiving coagulation replacement therapy) in single or multiple dosesfor a relatively short period, for example one to two weeks.

In practicing embodiments of the invention, one or more therapeuticallyeffective cycles of treatment will be administered to a subject. By“therapeutically effective cycle of treatment” is meant a cycle oftreatment that when administered, brings about the desired therapeuticresponse with respect to treatment. For example, one or moretherapeutically effective cycles of treatment may increase the rate ofblood clotting as determined by blood clotting assays (e.g., CAT, aPTT,described in detail below) by 1% or more, such as 5% or more, such as10% or more, such as 15% or more, such as 20% or more, such as 30% ormore, such as 40% or more, such as 50% or more, such as 75% or more,such as 90% or more, such as 95% or more, including increasing the rateof blood clot formation by 99% or more. In other instances, one or moretherapeutically effective cycles of treatment may increase the rate ofblood clot formation by 1.5-fold or more, such as 2-fold or more, suchas 5-fold or more, such as 10-fold or more, such as 50-fold or more,including increasing the rate of blood clot formation by 100-fold ormore. In some embodiments, subjects treated by methods of the inventionexhibit a positive therapeutic response. By “positive therapeuticresponse” is meant that the subject exhibits an improvement in one ormore symptoms of a bleeding disorder. For example, a subject exhibitinga positive therapeutic response to methods provided by the invention mayinclude but is not limited to responses such as shortened blood clottingtimes, reduced bleeding, reduced need for factor replacement therapy ora combination thereof. In certain embodiments, more than onetherapeutically effective cycle of treatment is administered.

As reviewed above, in practicing methods according to certainembodiments, a composition having an amount of a NASP is administered toa subject to enhance blood coagulation in the subject. Any convenientmode of administration may be employed. Modes of administration mayinclude, but are not limited to oral administration, injection (e.g.,subcutaneously, intravenously or intramuscularly), intravenous infusion,pulmonary application, rectal application, transdermal application,transmucosal application, intrathecal application, pericardialapplication, intra-arterial application, intracerebral application,intraocular application, intraperitoneal application or local (i.e.,direct) application. As discussed in greater detail below,pharmaceutical compositions of the invention may be in the form of aliquid solution or suspension, syrup, cream, ointment, tablet, capsule,powder, gel, matrix, suppository, or any combination thereof. Where acomposition having an amount of a NASP is administered in combinationwith a blood coagulation factor, as discussed in detail above, the modeof administration of each component may be the same or different. Forexample, in some instances, the composition having an amount of a NASPmay be locally applied (e.g., as a cream), whereas the blood coagulationfactor may be administered orally. In other instances, both thecomposition having an amount of a NASP and the blood coagulation factorare locally applied. In certain embodiments, a composition having anamount of a NASP may be used for localized administration, such as forexample, for the treatment of bleeding as a result of a lesion, injury,or surgery. In some instances, a NASP may be administered by injectionat the site of bleeding or in the form of a solid, liquid, or ointment,or applied by an adhesive tape.

In certain embodiments, methods of the invention provide foradministering a composition having an amount of a NASP prophylactically,such as for example before planned surgery. The composition may beapplied prophylactically as desired, such as one hour or more prior to aplanned procedure, such as 10 hours prior to a planned procedure, suchas 24 hours prior to a planned procedure, and including one week priorto a planned procedure. In some instances, the composition administeredprior to or during a planned procedure may be a sustained-releaseformulation (e.g., transdermal patch, miniature implantable pumps,sustained release caplets or tablets), as described in greater detailbelow.

In certain embodiments, compositions of the invention can beadministered prior to, concurrent with, or subsequent to other agentsfor treating related or unrelated conditions. If provided at the sametime as other agents, compositions of the invention can be provided inthe same or in a different composition. Thus, NASPs of interest andother agents can be presented to the individual by way of concurrenttherapy. By “concurrent therapy” is intended administration to a subjectsuch that the therapeutic effect of the combination of the substances iscaused in the subject undergoing therapy. For example, concurrenttherapy may be achieved by administering compositions of the inventionand a pharmaceutical composition having at least one other agent, suchas a hemostatic agent or coagulation factor (e.g. FVIII or FIX), whichin combination comprise a therapeutically effective dose, according to aparticular dosing regimen. Similarly, one or more NASPs and therapeuticagents can be administered in at least one therapeutic dose.Administration of the separate pharmaceutical compositions can beperformed simultaneously or at different times (i.e., sequentially, ineither order, on the same day, or on different days), so long as thetherapeutic effect of the combination of these substances is caused inthe subject undergoing therapy.

Compositions

Aspects of the invention also include compositions for enhancing bloodcoagulation in a subject. In embodiments of the invention, compositionsinclude a combination of a NASP and a blood coagulation factor. NASPsfor use in the methods of the invention are sulfated polysaccharidesthat demonstrate procoagulant activity. The non-anticoagulant propertiesof potential NASPs may be determined using any of the clotting assaysdescribed herein, including calibrated automated thrombography (CAT) inFactor VIII and/or Factor IX deficient plasma, dilute prothrombin time(dPT) or activated partial thromboplastin time (aPTT) clotting assays.One measure of noncoagulant activity is to compare the NASP in questionwith the known anticoagulant heparin. For example, NASPs may exhibitone-third or less, such as one-tenth or less of the anticoagulantactivity (measured by statistically significant increase in clottingtime) of unfractionated heparin (MW range 8,000 to 30,000; mean 18,000Daltons). Thus, a NASP can demonstrate at least a two-fold loweranticoagulant activity as compared to heparin, such as a two- tofive-fold or lower anticoagulant activity as compared to heparin, andincluding a two- to 10-fold or lower anticoagulant activity as comparedto heparin, using any of the various clotting assays detailed herein.

In some embodiments, compositions of the invention include a NASP havinga sulfur content of 8% sulfur or more by weight and a blood coagulationfactor. In certain embodiment, the composition has a NASP that has asulfur content that is 10% sulfur or more by weight, such as 15% sulfuror more by weight, such as 20% sulfur or more by weight, including 25%sulfur or more by weight. In other embodiments, compositions of theinvention have NASPs that contain an amount of sulfur that varies, forexample ranging from 5 to 25% sulfur by weight, such as 5% to 20% sulfurby weight, such as 5 to 20% sulfur by weight, including 5 to 15% sulfurby weight.

As discussed above, the sulfur content of the NASPs may be present inthe form of sulfate. The overall amount of sulfate present in the NASPsmay vary. In certain embodiments, the overall amount of sulfate presentin NASPs of the invention is 20% sulfate or more by weight, such as 25%sulfate or more by weight, such as 35% sulfate or more by weight,including 50% sulfate or more by weight. In other embodiments, theoverall amount of sulfate in the NASPs ranges, for example from 5 to 50%sulfate by weight, such as 5 to 40% sulfate by weight, such as 5 to 30%sulfate by weight, such as 5 to 25% sulfate by weight, such as 10% to 25sulfate by weight, such as 10 to 20% sulfate by weight, including 10 to15% sulfate by weight.

Each polysaccharide residue in NASPs of the invention may have varyingdegrees of sulfation. As discussed above, by “degree of sulfation” ismeant the number of sulfate groups bonded to each saccharide residue onthe NASP polysaccharide backbone. In some embodiments, eachpolysaccharide residue (e.g., fucose, galactose, glucose, mannose,xylose as described in detail below) may contain one (i.e.,monosulfated) or more (polysulfated) sulfate moieties. For example, insome instances the saccharide residue may be sulfated at the 4-positionof the saccharide residue. In other instances the saccharide residue issulfated at the 3-position. In other instances the saccharide residue issulfated at the 2-position. In other instances, the saccharide residueis sulfated at the 6-position. In certain instances, the saccharideresidue may be sulfated at one or more of the 6-position, the4-position, the 3-position and the 2-position and any combinationsthereof. For example, the saccharide residue may be sulfated at the4-position and at the 3-position, or at the 4-position and at the2-position, or at the 3-position and the 2-position, or at the6-position, 3-position and the 2-position, etc. Each residue may haveidentical degrees of sulfation (e.g., all saccharide residues beingmonosulfated) or may have varying degrees of sulfation (e.g., somesaccharide residues having identical sulfation and some saccharideresidues having different sulfation). For example, 10% or more of thesaccharide residues of NASPs of the invention may be monosulfated, suchas 15% or more of the saccharide residues, such as 25% or more of thesaccharide residues, such as 50% or more of the saccharide residues,such as 75% or more of the saccharide residues, such as 90% or more ofthe saccharide residues, such as 95% or more of the saccharide residues,including 99% or more of the saccharide residues of NASPs of theinvention may be monosulfated. On the other hand, in some embodiments10% or more of the saccharide residues of NASPs of the invention arepolysulfated, such as 15% or more of the saccharide residues, such as25% or more of the saccharide residues, such as 50% or more of thesaccharide residues, such as 75% or more of the saccharide residues,such as 90% or more of the saccharide residues, such as 95% or more ofthe saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention may be polysulfated. Where bothmonosulfated and polysulfated saccharide residues are present, the ratioof monosulfated residues to polysulfated residues in NASPs of theinvention may vary, and in some instances may range between 1:1 and1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the molarratio of monosulfated residues to polysulfated residues (i.e.,monosulfated saccharide residues:polysulfated saccharide residues) inNASPs of interest may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In someembodiments, the ratio of polysulfated residues to monosulfated residues(i.e., polysulfated saccharide residues:monosulfated saccharideresidues) in the NASPs ranges between 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150;1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, ora range thereof. For example, the ratio of polysulfated saccharideresidues to monosulfated residues in NASPs of interest may range between1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and1:500; or 1:100 and 1:1000.

In some embodiments, saccharide residues of NASPs of interest may besulfated at the 4-position. In other embodiments, the saccharideresidues are sulfated at the 3-position. In certain embodiments, thesaccharide residues are sulfated at the 4-position and at the3-position. In other instances the saccharide residue is sulfated at the2-position. In other instances, the saccharide residue is sulfated atthe 6-position. In certain instances, the saccharide residue may besulfated one or more of the 6-position, the 4-position, the 3-positionand the 2-position and any combinations thereof. For example, 10% ormore of the saccharide residues of NASPs of the invention may besulfated at only one of the 6-position, 4-position, 3-position or at the2-position such as 15% or more of the saccharide residues, such as 25%or more of the saccharide residues, such as 50% or more of thesaccharide residues, such as 75% or more of the saccharide residues,such as 90% or more of the saccharide residues, such as 95% or more ofthe saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention may be sulfated at only one of the6-position, 4-position, 3-position or at the 2-position. In otherembodiments 10% or more of the saccharide residues of NASPs of theinvention are sulfated at more than one of the 6-position, 4-position,3-position and at the 2-position, such as 15% or more of the saccharideresidues, such as 25% or more of the saccharide residues, such as 50% ormore of the saccharide residues, such as 75% or more of the saccharideresidues, such as 90% or more of the saccharide residues, such as 95% ormore of the saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention are sulfated at more than one of the6-position, 4-position, 3-position or at the 2-position. In certainembodiments 10% or more of the saccharide residues of NASPs of theinvention are sulfated at both the 3-position and the 4-position, suchas 15% or more of the saccharide residues, such as 25% or more of thesaccharide residues, such as 50% or more of the saccharide residues,such as 75% or more of the saccharide residues, such as 90% or more ofthe saccharide residues, such as 95% or more of the saccharide residues,including 99% or more of the saccharide residues of NASPs of theinvention are sulfated at both the 3-position and the 4-position. Whereboth saccharide residues sulfated at the 4-position and saccharideresidues sulfated at the 3-position are present, the ratio of saccharideresidues sulfated at the 4-position to saccharide residues sulfated atthe 3-position may vary, and in some instances may range between 1:1 and1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the molarratio of saccharide residues sulfated at the 4-position to saccharideresidues sulfated at the 3-position in NASPs of interest may rangebetween 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50and 1:500; or 1:100 and 1:1000. In some embodiments, the ratio ofsaccharide residues sulfated at the 3-position to saccharide residuessulfated at the 4-position in the NASPs ranges between 1:1 and 1:2.5;1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the ratio ofsaccharide residues sulfated at the 3-position to saccharide residuessulfated at the 4-position in NASPs of interest may range between 1:1and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500;or 1:100 and 1:1000. Any convenient protocol can be employed todetermine the type of sulfated saccharide residues of the NASPs, such asdescribed above.

As described in detail above, NASPs of the invention may be extractedfrom a biological source. In some instances NASPs of interest arefucoidans having a sulfur content of 8% sulfur or more by weight. Incertain embodiments, fucoidans having a sulfur content of 8% sulfur ormore by weight include, but are not limited to, Fucoidan GFS 5508005,Undaria pinnatifida, depyrogenated; Fucoidan GFS 5508004, Undariapinnatifida; Fucoidan GFS 5508003, Undaria pinnatifida; Fucoidan5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucusvesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5kD; Fucoidan 5308004, Fucus vesiculosus; Fucoidan 5308005, Fucusvesiculosus; Fucoidan L/FVF1091, Fucus vesiculosus; Fucoidan VG201096A,Fucus vesiculosus; Fucoidan VG201096B, Fucus vesiculosus; Fucoidan VG57,Undaria pinnatifida, high charge (high sulphation, deacetylated);Fucoidan VG50, Ascophyllum nodosum, max. MW peak 149.7 kD; and anycombinations thereof.

In addition, compositions of the invention also include one or moreblood coagulation factors. For example, compositions of the inventionmay include an amount of one or more NASPs in combination with one ormore blood coagulation factors. Blood coagulation factors of interestinclude, but are not limited to factor XI, factor XII, prekallikrein,high molecular weight kininogen (HMWK), factor V, factor VII, factorVIII, factor IX, factor X, factor XIII, factor II, factor VIIa, and vonWillebrands factor, factor Xa, factor IXa, factor XIa, factor XIIa, andVIIIa, prekallekrein, and high-molecular weight kininogen, tissuefactor, factor VIIa, factor Va, and factor Xa.

The amount (i.e., mass) of each of the NASP and blood coagulation factorin compositions of the invention may vary, ranging from 0.001 mg to 1000mg, such as 0.01 mg to 500 mg, such as 0.1 mg to 250 mg, such as 0.5 mgto 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg. As such, incompositions of the invention, the mass ratio of the NASP having asulfur content that is 8% sulfur or more by weight to blood coagulationfactor may vary, and in some instances may range between 1:1 and 1:2.5;1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratioof the NASP having a sulfur content that is 8% sulfur or more by weightto blood coagulation factor may range between 1:1 and 1:10; 1:5 and1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and1:1000. In some embodiments, the mass ratio of the blood coagulationfactor to the NASP having a sulfur content that is 8% sulfur or more byweight ranges between 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a rangethereof. For example, the mass ratio of the blood coagulation factor tothe composition that contains a NASP having a sulfur content that is 8%sulfur or more by weight may range between 1:1 and 1:10; 1:5 and 1:25;1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

In other embodiments, compositions of the invention include a NASPhaving 40% or more fucose saccharide residues and a blood coagulationfactor. As described above, the saccharide content of NASPs may vary. Insome instances, the saccharide content of NASPs of interest may include,but is not limited to fucose residues, xylose residues, galactoseresidues, glucose residues, mannose residues, rhamnose residues,arabinose residues and uronic acid. In some embodiments, NASPs ofinterest are composed of two or more of fucose residues, xyloseresidues, galactose residues, glucose residues, mannose residues,rhamnose residues, arabinose residues and uronic acid. The amount ofeach saccharide residue in NASPs of interest may vary. For example, 40%or more of the saccharide residues of NASPs of the invention may befucose saccharide residues, such as 45% or more of the saccharideresidues, such as 50% or more of the saccharide residues, such as 55% ormore of the saccharide residues, such as 65% or more of the saccharideresidues, such as 75% or more of the saccharide residues, such as 90% ormore of the saccharide residues, including 99% or more of the saccharideresidues of NASPs of the invention may be fucose saccharide residues. Inother instances, 1% or more of the saccharide residues of NASPs of theinvention may be galactose saccharide residues, such as 5% or more ofthe saccharide residues, such as 10% or more of the saccharide residues,such as 15% or more of the saccharide residues, such as 20% or more ofthe saccharide residues, including 25% or more of the saccharideresidues of NASPs of the invention may be galactose saccharide residues.In yet other instances, 1% or more of the saccharide residues of NASPsof the invention may be uronic acid saccharide residues, such as 5% ormore of the saccharide residues, such as 10% or more of the saccharideresidues, such as 15% or more of the saccharide residues, such as 20% ormore of the saccharide residues, including 25% or more of the saccharideresidues of NASPs of the invention may be uronic acid saccharideresidues.

In embodiments of the invention, NASPs of interest may be a linear(i.e., unbranched) polysaccharide or may be a branched polysaccharide.In certain instances, NASPs may have portions of its structure that islinear and other parts of its structure that is branched. As discussedabove, a linear polysaccharide is a polysaccharide or part of apolysaccharide that contains only α-1,4 glycosidic bonds or α-1,3glycosidic bonds, or alternating α-1,3/α-1,4 glycosidic bonds and abranched polysaccharide is a polysaccharide or part of a polysaccharidethat contains two or more glycosidic bonds to other saccharide residues,where one of the glycosidic bonds is an α-1,4-glycosidic bond or α-1,3glycosidic bonds, or alternating α-1,3/α-1,4 glycosidic bonds and theother is an α-1,6-glycosidic bond. The amount of branching in thestructure of NASPs of interest may vary.

In some embodiments, compositions of the invention include a NASP thatcontains 40% or more fucose saccharide residues. For example, NASPs ofinterest may contain 45% or more fucose saccharide residues, such as 50%or more fucose saccharide residues, such as 60% or more fucosesaccharide residues, such as 75% or more fucose saccharide residues,such as 85% or more fucose saccharide residues, such as 90% or morefucose saccharide residues, including 95% or more fucose saccharideresidues. In certain embodiments, NASPs of interest may contain 40% ormore sulfated esters of fucose saccharide residues, such as 50% or moresulfated esters of fucose saccharide residues, such as 60% or moresulfated esters of fucose saccharide residues, such as 75% or moresulfated esters of fucose saccharide residues, such as 85% or moresulfated esters of fucose saccharide residues, such as 90% or moresulfated esters of fucose saccharide residues, including 95% or moresulfated esters of fucose saccharide residues. As described in detailabove, sulfated esters of fuose saccharide residues may vary in theamount of sulfation, regio selectivity of sulfation as well as degree ofsulfation. For example, sulfated esters of fucose saccharide residuesmay, in some instances, be monosulfated. In other instances, sulfatedesters of fucose saccharide residues may be polysulfated. Likewise, incertain instances, sulfated esters of fucose saccharide residues may besulfated that the 4-position. On the other hand, sulfated esters offucose saccharide residues may be sulfated at the 3-position.

In certain embodiments, NASPs of interest contain 40% or more fucosesaccharide residues and 20% or more galactose saccharide residues, suchas 45% or more fucose saccharide residues and 20% or more galactoseresidues, such as 50% or more fucose saccharide residues and 20% or moregalactose residues, such as 60% or more fucose saccharide residues and20% or more galactose residues, such as 70% or more fucose saccharideresidues and 20% or more galactose residues. In other embodiments, NASPsof interest contain 40% or more fucose saccharide residues and 25% ormore galactose saccharide residues, such as 40% or more fucosesaccharide residues and 30% or more galactose saccharide residues, andincluding 40% or more fucose saccharide residues and 40% or moregalactose saccharides residues.

As described in detail above, NASPs of the invention may be extractedfrom a biological source. In some instances NASPs of interest arefucoidans that contain 40% or more fucose saccharide residues. Incertain embodiments, fucoidans of interest may include but are notlimited to Fucoidan GFS 5508005, Undaria pinnatifida, depyrogenated;Fucoidan GFS 5508004, Undaria pinnatifida; Fucoidan VG 23, E. Maxima;Fucoidan L/FVF1093, Fucus vesiculosus, Fucoidan L/FVF1092, Fucusvesiculosus; and any combinations thereof.

Compositions of the invention also include one or more blood coagulationfactors in addition to a NASP having 40% or more fucose saccharideresidues. For example, compositions of the invention may include anamount of one or more NASPs in combination with one or more bloodcoagulation factors. Blood coagulation factors of interest include, butare not limited to factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, factor VIIa, and von Willebrandsfactor, factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa,prekallekrein, and high-molecular weight kininogen, tissue factor,factor VIIa, factor Va, and factor Xa.

The amount (i.e, mass) of each of the NASPs and blood coagulation factorin compositions of the invention may vary, ranging from 0.001 mg to 1000mg, such as 0.01 mg to 500 mg, such as 0.1 mg to 250 mg, such as 0.5 mgto 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg. As such, incompositions of the invention, the mass ratio of the NASP having 40% ormore fucose saccharide residues to blood coagulation factor may vary,and in some instances may range between 1:1 and 1:2.5; 1:2.5 and 1:5;1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and1:1000, or a range thereof. For example, the mass ratio of the NASPhaving 40% or more fucose saccharide residues to blood coagulationfactor may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, themass ratio of the blood coagulation factor to the NASP having 40% ormore fucose saccharide residues ranges between 1:1 and 1:2.5; 1:2.5 and1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and1:1000, or a range thereof. For example, the mass ratio of the bloodcoagulation factor to the composition that contains a NASP having 40% ormore fucose saccharide residues may range between 1:1 and 1:10; 1:5 and1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and1:1000.

In certain embodiments, compositions of the invention include fucoidans.Fucoidans are naturally-occurring complex sulfated polysaccharidescompounds which may be extracted from certain edible seaweeds, brownalgae and echinoderms (e.g., sea urchins, sea cucumbers). As used hereinthe term, “fucoidan” refers to a diverse group of moieties extractedfrom a biological source of low sulfate polymers rather than a singlechemical entity. Fucoidan from various species of brown algae andechinoderm differ in the amount of fucose in their backbone, the degreeand pattern of sulfation, structure (linear versus branching), andproportions of individual saccharides and uronic acid.

Fucoidans for use in the present invention may be extracted, furtherpurified and/or modified from natural sources (e.g. brown algae).Fucoidans can be isolated from algae by hot water, by acid or ethanolextraction, or by enzymatic digestion, followed by isolation fromaqueous solution by precipitation (e.g., by addition of organicsolvents) or ultrafiltered.

Fucoidans in the present invention may be extracted from organisms fromthe genus Fucus, Laminaria, Cladosiphon, Namacystus, Undaria, Chordaria,Sargassum, Leathesia, Desmarestia, Dictyomphon, Dictyota, Padina,Spatoglossum, Adenocystis, Pylayella, Ascophyllum, Bifurcaria,Himanthalia, Hizikia, Pelvetia, Alaria, Arthrothamnus, Chorda, Ecklonia,Eisenia, Macrocystis, Nereocystis, Petalonia, Scytosiphon, andSaundersella, among others.

Fucoidans described herein may be heterogeneous mixtures of fucoidansvarying in sulfur content, degree of sulfation, saccharide content andmolecular weight.

Fucoidans of interest may range in average molecular weight from about10 daltons to about 500,000 daltons, such as from about 100 daltons toabout 300,000 daltons, such as from 1000 daltons to 250,000 daltons,including 1000 daltons to 150,000 daltons. Molecular weights of fucoidancan be determined by any convenient protocol, such as for example, gelpermeation chromatography or high-performance size-exclusionchromatography (HPSEC), capillary electrophoresis, PAGE (polyacrylamidegel electrophoresis), agarose gel electrophoresis, among others.

In some embodiments, fucoidans of interest may be heterogeneous mixturesof sulfated polysaccharides having varying molecular weights. Forexample, in some instances, 5% or more of the fucoidan composition has amolecular weight that ranges from 10 to 30,000 daltons, such as 10% ormore, such as 25% or more, such as 50% or more, such as 75% or more,such as 90% or more, including 95% or more of the fucoidan compositionhas a molecular weight that ranges from 10 to 30,000 daltons. In otherembodiments, 5% or more of the fucoidan composition has a molecularweight that ranges from 30,000 daltons to 75,000 daltons, such as 10% ormore, such as 25% or more, such as 50% or more, such as 75% or more,such as 90% or more, including 95% or more of the fucoidan compositionhas a molecular weight that ranges from 30,000 to 75,000 daltons. In yetother embodiments, 5% or more of the fucoidan composition has amolecular weight that are greater than 75,000 daltons, such as 10% ormore, such as 25% or more, such as 50% or more, such as 75% or more,such as 90% or more, including 95% or more of the fucoidan compositionhas a molecular weight that is greater than 75,000 daltons.

In certain embodiments, low molecular weight fucoidans may be employedfor enhancing blood coagulation as provided by methods and compositionsof the invention. By “low molecular weight fucoidan” is meant a fucoidanhaving a weight average molecular weight that ranges from about 10 to30,000 daltons, such as for example 100 to 30,000 daltons, such as 500to 25,000 daltons, including 1000 to 15,000 daltons. Examples of lowmolecular weight fucoidans may include, but are not limited to naturallyoccurring fucoidans having a molecular weight ranging from 10 to 30,000daltons, fragments of larger molecular weight fucoidans produced by acidor enzyme hydrolysis of the larger molecular weight fucoidan, or may beisolated fractions having molecular weights ranging from 10 to 30,000daltons from a fractionated fucoidan sample.

In some embodiments, fucoidans extracted from a biological source may befractionated to isolate low molecular weight fucoidans (i.e., fractionscontaining fucoidans having molecular weight ranging from 10-30,000daltons). Any convenient protocol may be used to fractionate fucoidansof interest, including but not limited to size exclusion chromotagraphy,gel permeation chromotagraphy, capillary electrophoresis, among others.

In certain instances, low molecular weight fucoidans obtained byfractionating a fucoidan sample may be employed for enhancing bloodcoagulation as provided by the methods and compositions of theinvention. For example, fucoidans extracted from a biological source maybe fractionated to isolate fucoidans having molecular weights that rangefrom 10 to 30,000 daltons, such as 10 to 5000 daltons, such as 5000 to10,000 daltons, such as 10,000 to 15,000 daltons, and including 15,000to 30,000 daltons. In certain embodiments, one or more of thesefractions may be administered for enhancing blood coagulation in asubject, such as by the methods described above.

In certain embodiments, different molecular weight fractions may beprepared by acid-hydrolysis or radical depolymerization of highmolecular weight fucoidan. The molecular weight ranges of the resultingproducts may be adjusted based upon the stringency of the hydrolysis ordepolymerization conditions employed. Fractions may then be furtherpurified using ion exchange chromatography. For instance, to obtainmiddle and low molecular weight fractions of fucoidan, high molecularweight fucoidan may be hydrolyzed using an acid such as HCl (or anyother suitable acid) at concentrations ranging from 0.02 to 1.5 M and attemperatures ranging from 25° C. to 80° C. Hydrolysis reaction timeswill typically range from 15 minutes to several hours. The resultinghydrolyzed reaction mixture is then neutralized by addition of base(e.g., sodium hydroxide). Salts are subsequently removed, for example,by electrodialysis, and the hydrolysis products are analyzed todetermine weight average molecular weight, saccharide content, andsulfur content, using conventional analytical techniques forcarbohydrate analysis. Alternatively, enzymatic methods may be employedto degrade fucoidans using, e.g., glycosidases such as fucan sulfatehydrolase (fucoidanase EC 3.2.1.44) and α-L-fucosidase EC 3.2.1.51.Fucoidans for use in the invention may be heterogeneous or homogeneous,depending upon the degree of separation employed.

In certain embodiments, compositions of the invention include a bloodcoagulation factor in combination with a fucoidan extracted from abiological source, such as for example, Fucoidan 5307002, Fucusvesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucus vesiculosus,hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5 kD; FucoidanVG57, Undaria pinnatifida, high charge (high sulfation, deacetylated);Fucoidan GFS (5508005), Undaria pinnatifida, depyrogenated; Fucoidan GFS(L/FVF-01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD;Fucoidan GFS (L/FVF-01092), Fucus vesiculosus, depyrogenated, max. MWpeak 260 kD; Fucoidan GFS (L/FVF-01093), Fucus vesiculosus, hydrolyzeddepyrogenated, max. MW peak 36 kD; Maritech® Ecklonia radiata extract;Maritech® Ecklonia maxima extract; Maritech® Macrocystis pyriferaextract; Maritech® Immune trial Fucoidan Blend; and any combinationsthereof.

As described above, fucoidans of interest may be extracted from abiological source. In some instances, crude fucoidan compositionsextracted from a biological source may also contain impurities. By“impurities” is meant any component of the crude fucoidan compositionwhich may be undesirable or is detrimental to the fucoidan composition.For example, impurities may interfere (i.e., diminish) or inhibit aparticular desirable property of fucoidans of the invention, such as forexample procoagulant activity. In other embodiments, impurities may notbe detrimental to the function of the fucoidan composition, but mayresult in the fucoidan composition being unsuitable for administrationto a subject, such as for example containing elevated levels of toxins,bacteria content or high levels of trace metal ions (e.g., arsenic,lead, cadmium or mercury) as described below. Impurities may include,but are not limited to residual moisture, protein, endotoxins, alginate,uronic acids, trace elements and metal ions.

The amount of protein impurities present in extracted fucoidancompositions of the invention may vary, ranging from 0.2% to 6% byweight, such as 0.25% to 5% by weight, such as 0.5% to 2.5% by weight,including 1.0% to 2.0% by weight. Further, endotoxin levels in extractedfucoidan compositions may also vary, ranging from 0.1 EU/mg to 75 EU/mg,such as 0.5 EU/mg to 50 EU/mg, such as 1 EU/mg to 25 EU/mg, including 5EU/mg to 10 EU/mg. The residual moisture content of extracted fucoidancompositions of the invention may also vary, ranging from 5% to 20%,such as 5% to 15%, including 5% to 10%.

In some embodiments, impurities may include uronic acids. Uronic acidsmay be present in extracted fucoidan compositions of the invention in anamount that varies, ranging from 1% to 60% by weight, such as 5% to 50%by weight, such as 10% to 40% by weight, and including 15% to 25% byweight. Uronic acid impurities may be detected and quantified using anyconvenient protocol, such as for example, Carbazole Assay or nuclearmagnetic resonance spectroscopy.

In some embodiments, impurities may include trace elements and metalions. Trace elements and metal ions may include, but are not limited toaluminum, arsenic, bromine, cadmium, cerium, chromium, cobalt, iodine,lead, lithium, manganese, mercury, molybdenum, nickel, phosphorus,rubidium, tin, tungsten, uranium, vanadium. Trace elements and metalions (e.g., As, Cd Hg, Pb) may be present in extracted fucoidancompositions of the invention in an amount that varies, ranging from0.05 μg/g to 3.0 μg/g, such as 0.1 μg/g to 2.5 μg/g, such as 0.25 μg/gto 2.0 μg/g, and including 0.5 μg/g to 1.5 μg/g. Trace elements andmetal ions may be detected using any convenient protocol, such as forexample mass spectrometry, inductively coupled plasma, ionchromatography, gas chromatography, atomic absorption, graphite furnaceatomic absorption spectrometry, inductively coupled plasma massspectrometry, inductively coupled plasma atomic emission spectrometry,flame atomic absorption spectrometry, acidimetric titration, or anycombination thereof.

In some embodiments, fucoidan compositions extracted from a biologicalsource may be purified prior to administering to a subject. Impuritiesin fucoidan compositions may be purified using any convenient protocol.Methods for removing impurities and purifying a fucoidan compositionextracted from a biological source is described in detail in co-pendingU.S. patent application Ser. No. 12/449,712, filed Feb. 25, 2010, thedisclosure of which is herein incorporated by reference.

Blood coagulation factors which are administered in combination withNASPs of interest may include, but are not limited to factor XI, factorXII, prekallikrein, high molecular weight kininogen (HMWK), factor V,factor VII, factor VIII, factor IX, factor X, factor XIII, factor II,factor VIIa, and von Willebrands factor, factor Xa, factor IXa, factorXIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weightkininogen, tissue factor, factor VIIa, factor Va, and factor Xa.

The amount (i.e., mass) of each of the fucoidan and blood coagulationfactor in compositions of the invention may vary, ranging from 0.001 mgto 1000 mg, such as 0.01 mg to 500 mg, such as 0.1 mg to 250 mg, such as0.5 mg to 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg. Assuch, in compositions of the invention, the mass ratio of the fucoidanto blood coagulation factor may vary, and in some instances may rangebetween 1:1 and 1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. Forexample, the mass ratio of the fucoidan to blood coagulation factor mayrange between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100;1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratioof the blood coagulation factor to the fucoidan ranges between 1:1 and1:2.5; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratioof the blood coagulation factor to the fucoidan may range between 1:1and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500;or 1:100 and 1:1000.

Compositions of the invention may be homogeneous, containing only asingle type of NASP. In other embodiments, compositions of interest areheterogenous mixtures of two or more NASPs. For example, heterogenousmixtures may contain two or more NASPs that vary with respect tomonosaccharide content, sulfur content, degree of sulfation as well asNASPs having heterogenous or homogeneous distributions of molecularweight. In some instances, compositions of the invention are fucoidansthat have low molecular weight. In other instances, compositions of theinvention are composed of fucoidans having a broad range of molecularweight.

In certain embodiments, compositions of the invention may furtherinclude one or more pharmaceutically acceptable excipients as part of apharmaceutical composition. Excipients may include, but are not limitedto, carbohydrates, inorganic salts, antimicrobial agents, antioxidants,surfactants, buffers, acids, bases, and any combinations thereof.Excipients suitable for injectable compositions may include water,alcohols, polyols, glycerine, vegetable oils, phospholipids, andsurfactants. A carbohydrate such as a sugar, a derivatized sugar such asan alditol, aldonic acid, an esterified sugar, and/or a sugar polymermay also be employed. Some carbohydrate excipients of interest include,for example, monosaccharides, such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and thelike. Inorganic salts may include, but are not limited to citric acid,sodium chloride, potassium chloride, sodium sulfate, potassium nitrate,sodium phosphate monobasic, sodium phosphate dibasic, and anycombinations thereof.

In certain embodiments, compositions of the invention may also includean antimicrobial agent for preventing or deterring microbial growth,such as for example benzalkonium chloride, benzethonium chloride, benzylalcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethylalcohol, phenylmercuric nitrate, thimersol, and any combinationsthereof.

One or more antioxidants may also be employed. Antioxidants, which canreduce or prevent oxidation and thus deterioration of the composition,may include, for example, ascorbyl palmitate, butylated hydroxyanisole,butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite, and any combinations thereof.

One or more surfactants may also be included in compositions of theinvention. For example, suitable surfactants may include, but are notlimited to polysorbates, such as “Tween 20” and “Tween 80,” andpluronics such as F68 and F88 (BASF, Mount Olive, N.J.); sorbitanesters; lipids, such as phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines (although preferably notin liposomal form), fatty acids and fatty esters; steroids, such ascholesterol; chelating agents, such as EDTA; and zinc and other cations.

Acids or bases may also be present in compositions of the invention. Forexample, acids may include but are not limited to hydrochloric acid,acetic acid, phosphoric acid, citric acid, malic acid, lactic acid,formic acid, trichloroacetic acid, nitric acid, perchloric acid,phosphoric acid, sulfuric acid, fumaric acid, and any combinationsthereof. Examples bases include, but are not limited to sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and any combinations thereof.

The amount of any individual excipient in the composition will varydepending on the nature and function of the excipient and particularneeds of the composition. Typically, the optimal amount of anyindividual excipient is determined through routine experimentation,i.e., by preparing compositions containing varying amounts of theexcipient (ranging from low to high), examining the stability and otherparameters, and then determining the range at which optimal performanceis attained with no significant adverse effects. Generally, however, theexcipient(s) will be present in the composition in an amount of about 1%to about 99% by weight, such as from about 5% to about 98% by weight,such as from about 15 to about 95% by weight of the excipient, includingless than 30% by weight. Pharmaceutical excipients along with otherexcipients that may be employed in compositions of the invention aredescribed in “Remington: The Science & Practice of Pharmacy”, 19th ed.,Williams & Williams, (1995), the “Physician's Desk Reference”, 52nd ed.,Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook ofPharmaceutical Excipients, 3rd Edition, American PharmaceuticalAssociation, Washington, D.C., 2000, the disclosure of which is hereinincorporated by reference.

As described above, compositions of the invention may be administered byany convenient mode of administration. As such, the formulation mayvary. For example, compositions of the invention may be an injection,e.g., powders or lyophilates that can be reconstituted with a solventprior to use, as well as ready for injection solutions or suspensions,dry insoluble compositions for combination with a vehicle prior to use,and emulsions and liquid concentrates for dilution prior toadministration. In embodiments where compositions of the invention areemployed for injections, diluents for reconstituting solid compositionsprior to injection may include, but is not limited to bacteriostaticwater for injection, dextrose 5% in water, phosphate buffered saline,Ringer's solution, saline, sterile water, deionized water, and anycombinations thereof. In some embodiments, pharmaceutical compositionsof the invention may be in the form of a liquid solution or suspension,syrup, cream, ointment, tablet, capsule, powder, gel, matrix,suppository, or any combination thereof.

Compositions of the invention may be pre-loaded into a syringe, animplantation device, or the like, depending upon the intended mode ofdelivery and use. In certain embodiments, the compositions are in unitdosage form, such that an amount of the composition is ready in a singledose, in a premeasured or pre-packaged form.

Utility

The subject methods and compositions find use in any situation wherethere is a desire to enhance blood coagulation in a subject and thesubject is responsive to treatment with a NASP. In certain embodiments,the subject methods and compositions may be employed to treat bleedingdisorders, such as a chronic or acute bleeding disorder, a congenitalcoagulation disorder caused by a blood factor deficiency, an acquiredcoagulation disorder and administration of an anticoagulant. Forexample, bleeding disorders may include, but are not limited tohemophilia A, hemophilia B, von Willebrand disease, idiopathicthrombocytopenia, a deficiency of one or more contact factors, such asFactor XI, Factor XII, prekallikrein, and high molecular weightkininogen (HMWK), a deficiency of one or more factors associated withclinically significant bleeding, such as Factor V, Factor VII, FactorVIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia),and von Willebrands factor, a vitamin K deficiency, a disorder offibrinogen, including afibrinogenemia, hypofibrinogenemia, anddysfibrinogenemia, an alpha₂-antiplasmin deficiency, and excessivebleeding such as caused by liver disease, renal disease,thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage,hemarthroses, surgery, trauma, hypothermia, menstruation, and pregnancy.

The subject methods and compositions also find use in enhancing bloodcoagulation to treat a congenital coagulation disorder or an acquiredcoagulation disorder caused by a blood factor deficiency. The bloodfactor deficiency may be caused by deficiencies of one or more factors,including but not limited to, factor V, factor VII, factor VIII, factorIX, factor XI, factor XII, factor XIII, and von Willebrand factor.

The subject methods and compositions also find use in enhancing bloodcoagulation in order to improve hemostasis in treating bleedingdisorders, such as those associated with deficiencies of coagulationfactors or for reversing the effects of anticoagulants in a subject. Forexample, enhancing blood coagulation by methods and compositions of theinvention may be employed to to treat bleeding disorders such ascongenital coagulation disorders, acquired coagulation disorders, andhemorrhagic conditions induced by trauma. Examples of bleeding disordersthat may be treated with NASPs include, but are not limited to,hemophilia A, hemophilia B, von Willebrand disease, idiopathicthrombocytopenia, a deficiency of one or more contact factors, such asFactor XI, Factor XII, prekallikrein, and high molecular weightkininogen (HMWK), a deficiency of one or more factors associated withclinically significant bleeding, such as Factor V, Factor VII, FactorVIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia),and von Willebrands factor, a vitamin K deficiency, a disorder offibrinogen, including afibrinogenemia, hypofibrinogenemia, anddysfibrinogenemia, an alpha₂-antiplasmin deficiency, and excessivebleeding such as caused by liver disease, renal disease,thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage,hemarthroses, surgery, trauma, hypothermia, menstruation, and pregnancy.In certain embodiments, methods and compositions of the invention areused to treat congenital coagulation disorders including hemophilia A,hemophilia B, and von Willebrands disease. In other embodiments, NASPsare used to treat acquired coagulation disorders, including deficienciesof factor VIII, von Willebrand factor, factor IX, factor V, factor XI,factor XII and factor XIII, particularly disorders caused by inhibitorsor autoimmunity against blood coagulation factors, or haemostaticdisorders caused by a disease or condition that results in reducedsynthesis of coagulation factors.

In some embodiments, the bleeding disorder may be a chronic condition(e.g., a congenital or acquired coagulation factor deficiency) requiringthe subject methods and compositions in multiple doses over an extendedperiod. Alternatively, methods and compositions of the invention may beadministered to treat an acute condition (e.g., bleeding caused bysurgery or trauma, or factor inhibitor/autoimmune episodes in subjectsreceiving coagulation replacement therapy) in single or multiple dosesfor a relatively short period, for example one to two weeks.

The subject methods and compositions also find use in enhancing bloodcoagulation in a subject undergoing a surgical or invasive procedure.

The subject methods and compositions also find use in enhancing bloodcoagulation in order to reverse the effects of an anticoagulant in asubject, the method comprising administering a therapeutically effectiveamount of a composition comprising a NASP to the subject. In certainembodiments, the subject may have been treated with an anticoagulantincluding, but not limited to, heparin, a coumarin derivative, such aswarfarin or dicumarol, TFPI, AT III, lupus anticoagulant, nematodeanticoagulant peptide (NAPc2), active-site blocked factor VIIa (factorVIIai), factor IXa inhibitors, factor Xa inhibitors, includingfondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906), inhibitorsof factors Va and VIIIa, including activated protein C (APC) and solublethrombomodulin, thrombin inhibitors, including hirudin, bivalirudin,argatroban, and ximelagatran. In certain embodiments, the anticoagulantin the subject may be an antibody that binds a clotting factor,including but not limited to, an antibody that binds to Factor V, FactorVII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II, FactorXI, Factor XII, von Willebrands factor, prekallikrein, or high molecularweight kininogen (HMWK).

In another aspect, the invention provides a method for treating asubject undergoing a surgical or invasive procedure wherein improvedblood clotting would be desirable, comprising administering atherapeutically effective amount of a composition comprising a NASP asdetailed herein to the subject. In certain embodiments, the NASP can becoadministered with one or more different NASPs and/or in combinationwith one or more other therapeutic agents to the subject undergoing asurgical or invasive procedure. For example, the subject may beadministered a therapeutically effective amount of one or more factorsselected from the group consisting of factor XI, factor XII,prekallikrein, high molecular weight kininogen (HMWK), factor V, factorVII, factor VIII, factor IX, factor X, factor XIII, factor II, factorVIIa, and von Willebrands factor. Treatment may further compriseadministering a procoagulant, such as an activator of the intrinsiccoagulation pathway, including factor Xa, factor IXa, factor XIa, factorXIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; oran activator of the extrinsic coagulation pathway, including tissuefactor, factor VIIa, factor Va, and factor Xa. Therapeutic agents usedto treat a subject undergoing a surgical or invasive procedure can beadministered in the same or different compositions and concurrently,before, or after administration of the NASP.

In another aspect, the invention provides a method of measuringacceleration of clotting by a NASP as detailed herein in a biologicalsample, the method including: combining the biological sample withcompositions of the invention; measuring the clotting time of thebiological sample; comparing the clotting time of the biological sampleto the clotting time of a corresponding biological sample not exposed tocompositions of the invention, wherein a decrease in the clotting timeof the biological sample exposed to the NASP, if observed, is indicativeof a NASP that accelerates clotting.

As disclosed above, hemostatic agents, blood factors, and medicationsmay also be employed. For example, the subject may be administered oneor more blood coagulation factors such as factor XI, factor XII,prekallikrein, high molecular weight kininogen (HMWK), factor V, factorVII, factor VIII, factor IX, factor X, factor XIII, factor II, factorVIIa, von Willebrands factor, factor Xa, factor IXa, factor XIa, factorXIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen,tissue factor, factor VIIa, factor Va, and factor Xa.

Kits

Also provided are kits for use in practicing the subject methods, wherethe kits may include one or more of the above compositions, e.g., anNASP composition and/or blood coagulation factor, as described above.The kit may further include other components, e.g., administrationdevices, fluid sources, syringes, needles etc., which may find use inpracticing the subject methods. Various components may be packaged asdesired, e.g., together or separately.

In addition to above mentioned components, the subject kits may furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed, such as on paper or plastic, etc. As such,the instructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXPERIMENTAL

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Clotting Assays

The ability of NASPs to promote clotting and reduce bleeding isdetermined using various in vitro clotting assays (e.g., dPT and aPTTassays) and in vivo bleeding models (e.g. tail snip or cuticle bleedingtime determination in hemophilic mice or dogs). Clotting assays may beperformed in the presence of compositions of the invention, includingNASPs of interest and one or more blood factors, procoagulants, or otherreagents. For example, one or more factors can be added, include but arenot limited to, factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, and von Willebrands factor, tissuefactor, factor VIIa, factor Va, and factor Xa, factor IXa, factor XIa,factor XIIa, and VIIIa; and/or one or more reagents, including but notlimited to, APTT reagent, thromboplastin, fibrin, TFPI, Russell's vipervenom, micronized silica particles, ellagic acid, sulfatides, andkaolin.

Compositions of the present invention show anticoagulant activity onlyat concentrations significantly above the concentration at which theyexhibit procoagulant activity. The ratio of the concentration at whichundesired anticoagulant properties occur to the concentration at whichdesired procoagulant activities occur is referred to as the procoagulantindex. The procoagulant index for compositions of the present inventionmay be 5 or more, such as 10 or more, such as 30 or more, such as 100 ormore, such as 300 or more, and including 1000 or more.

Calibrated Automated Thrombography (CAT) Assay

In the CAT studies, the procoagulant activity of sulfatedpolysaccharides was examined in several plasmas from patients withcongenital coagulation factor deficiencies and FVIII-inhibited normalplasma, in order to study the procoagulant window. Pooled normal plasmaor plasmas from patients with congenital coagulation factor deficiencieswere obtained from George King, Bio-Medical Inc. Kansas USA. Accordingto the supplier, the residual coagulation factor activity for each ofthe coagulation factor deficient plasmas was lower than 1%. As a modelfor antibody mediated FVIII deficiency fresh frozen pooled normal plasma(George King, Bio-Medical Inc., Kansas, USA) was incubated with hightiter heat inactivated anti-human FVIII plasma raised in goat (4490BU/ml; Baxter BioScience, Vienna, Austria) giving rise to 50 BU/mL. Insome experiments, tissue factor pathway inhibitor (TFPI) activity wasblocked in presence or absence of the fucoidan by either a polyclonalgoat anti-human TFPI antibodies (R&D Systems, AF2974, Minneapolis, US)or a monoclonal anti-TFPI antibody directed against the positivelycharged C-terminus of TFPI (Sanquin White Label Products, MW 1848, cloneCLB/TFPI C-terminus, Amsterdam, The Netherlands) at plasma concentrationof 25 nM or 100 nM, respectively. If not indicated otherwise, theplasmas were mixed with corn trypsin inhibitor (CTI) (HematologicTechnologies, Inc., Essex Junction, Vt., USA), providing a finalconcentration of 40 μg/mL, for specific inhibition of factor XIIa.

Test samples were prepared by dissolving quantities of NASPs of interestin Hepes buffered saline and adding human serum albumin (Sigma-AldrichCorporation, St. Louis, Mo., USA) to a concentration of 5 mg/mL.Reference samples were prepared from reference proteins FVIII Immunate®reference standard (Baxter BioScience, Vienna, Austria); Factor eightinhibitor by-passing activity (FEIBA) reference standard (BaxterBioScience, Vienna, Austria); NovoSeven® recombinant activated FVII(Novo Nordisk A/S, Denmark) and purified human plasma FIX (EnzymeResearch Laboratories, South Bend, Ill., USA). A thrombin calibratorcompound was obtained from Thrombinoscope BV, Maastricht, TheNetherlands.

Activated Partial Thromboplastin Time (aPTT) Assay

The aPTT assay is performed as described previously with modifications.Detailed methods for the aPTT assay may be found in the PDR Staff.Physicians' Desk Reference. 2004, which is herein incorporated byreference. Briefly, 50 μL of thawed human plasma (normal or hemophilic)is added to test tubes. 5 μL of saline (e.g. Sigma) or 5 μl of testagent (e.g., NASP) dissolved in saline is mixed with 50 μl of plasma.aPTT reagent (e.g. STA APTT, Roche) is reconstituted in 5 ml distilledwater and 50 μL of the reconstituted solution containing the APTTreagent is added to each test tube and incubated for 2-3 minutes at 37°C. Afterwards 50 μL of 25 mM CaCl₂ is added to initiate clotting. Allpipetting steps and plasma clotting time measurements are carried outwith an ACL Elite Pro (Beckman Coulter) instrument. The experimentalsetup and mechanism of aPTT assay as presented herein is shown in FIG.3.

Dilute Prothrombin Time (dPT) Assay

One dPT assay for use herein is a modified standard clinical PT assay.Details methods for the dPT assay can be found in Nordfang et al. (1991)Thromb Haemost 66:464-467; Welsch et al. (1991) Thrombosis Research 64:213-222, which is herein incorporated by reference. A dilute prothrombintime assay with added tissue factor pathway inhibitor (TFPI-dPT) is usedto demonstrate the TFPI-inhibiting effect of fucoidan BAX513 inhemophilic patient plasma (George King Biomedical). Plasma samples arepre-incubated with 0.3 μg/mL full-length TFPI (aa 1-276, constitutivelyproduced by SKHep1) and BAX513 (0.03-1 μg/mL) for 15 min at RT. TFreagent TriniClot PT Excel S (Trinity Biotech), diluted inHepes-buffered saline 1:400 with 0.5% BSA is added to the plasma sampleson an ACL Pro Elite hemostasis analyzer (Instrumentation Laboratory).Clotting is initiated with 0.25 mM CaCl₂. The volume ratio ofplasma:TF:CaCl₂ was 1:1:1. The time for plasma clotting is measured witha ACL ProElite Hemostasis Analyzer.

Animal Bleeding Time Assays

The bleeding time assay can be used to measure changes in hemostasisfunction in normal or hemophilic (FVIII or FIX or vWF deficient) rodentsfollowing administration of a test agent (e.g., vehicle control orNASP). A test agent (e.g., vehicle control or NASP) is administered to arodent once or twice daily orally, parenterally, or by continuousinfusion. For example, 0.1 ml/10 g body weight (subscapular) of a testagent at a dose ranging from 0.1 to 10 mg/kg can be administered withsmall gauge needles twice a day for at least one day and preferably morethan 3 days. On the day bleeding time is assayed, rodents areanesthetized with ketamine/xylazine (or isoflurane). Rodents are linedup on a sterile pad with a petri dish of saline for tail immersion. EMLAcreme is applied to the tail of rodents at an intended cut site. Formice, the very tip of the tail is snipped, and the tail is placed intothe saline dish and a counter is started. For rats, an 8 mm long by 1 mmdeep incision is made on the dorsal part of the rat tail, which is thentransferred into saline. The time for cessation of visible bleeding intothe saline is recorded. For rodents, bleeding times are approximately 10minutes for normal control mice and 6 minutes for normal control rats.After completion of the bleeding time assay, the rodent's tail is driedwith sterile gauge, verified for hemostasis, and the rodent is returnedto the cage. Silver nitrate can be applied to the cut site if necessary.

Alternatively, bleeding times can be measured in mice (Broze et al.(2001) Thromb. Haemost. 85:747-748) or in dogs (Scallan et al. (2003)Blood 102:2031-2037; Pijnappels et al. (1986) Thromb. Haemost. 55:70-73)by other methods. Alternative or additional pharmacodynamic endpointsmay include sampling of blood from NASP-treated subjects for directanalysis or for plasma isolation, and measurement of ex vivo clottingtimes (e.g., Whole Blood Clotting Time and/or PT and/or APTT) orcoagulation factor levels.

Whole Blood Clotting Time (WBCT) Assay

The WBCT assay is performed as follows. Mice are briefly anesthetized inan isoflurane chamber. The mice are then bled (e.g. 150 μl) from theretro orbital plexus into plastic blood collection tubes. The tubes areplaced in a 37° C. water bath and a stop watch is used to measureclotting time. During this period, the tubes are inverted at 1 minuteintervals. The time required for blood clotting (full/not partial clot)is measured.

Example 2 Extraction and Purification of a Fucoidan Crude Composition

Methods for extracting and isolating fucoidans from edible seaweeds,brown algae and echinoderms (e.g., sea urchins, sea cucumbers) have beendescribed in detail in co-pending U.S. patent application Ser. No.12/449,712, filed Feb. 25, 2010, the disclosure of which is hereinincorporated by reference. Examples of NASPs and fucoidans of interestare listed in Table 1, below.

Crude fucoidan Laminaria japonica fucoidan extract (SIGMA) was purifiedas follows in order to reduce levels of heavy metals and proteins. Waterwas added to the crude fucoidan material at 40°-45° C. EDTA was thenadded to remove heavy metals and the pH was adjusted to 6.0. The mixturewas stirred for one hour and NaCl was added, the pH was adjusted to 6.0and the mixture stirred for 30 minutes. Absolute ethanol was addedslowly over 75 minutes at 20°-25° C. and the mixture was centrifuged atroom temperature to precipitate fucoidan. The supernatant was discardedand the precipitant dissolved with water at 40°-45° C. The NaCladdition, ethanol addition, centrifugation and precipitation steps werecarried out two more times (three times total). The pH of the finalsolution in water was adjusted to 6.0±0.2 and the solution cooled to20°-25° C. The solution was filtered through a 2 μm filter twice andonce through a 0.2 μm Posidyne filter into a sterile polyethylene bag.The filtrate was assayed for total neutral sugar content, a measure ofthe concentration of active fucoidan. This solution may be held at 2°-8°C. for up to a week. In order to lyophilize fucoidan, the purifiedsolution was filtered through a 0.2 μm filter into lyophilizer bags andplaced in the lyophilizer. The temperature was dropped to −40° C. andafter a vacuum was applied (<200 microns), the temperature was raisedand held at 10° C. for 4 hours, 20° C. for 20 hours, 25° C. for 24hours, and 40° C. for 24 hours. After the vacuum was released, the bagscontaining the lyophilized material were transferred to a tared30-gallon drum lined with a doubled polyethylene bag and sealed with alid and locking ring. The container was transferred to a weighing roomwhere the lyophilization bags were cut open and the contents emptiedinto the double-bag-lined drum. After the drum was weighed, the drumbags were sealed, desiccant was placed around the bags, and the drum wasresealed.

In the final packaging step, the lyophilized drug substance wastransferred from the drum to doubled polyethylene bags containing 1-500grams per bag. The bags were closed and packaged with desiccant intocontainers fixed with tamper-evident seals. The sealed containers werestored at 2-8° C.

TABLE 1 List of Compounds 1. Fucoidan 5307002, Fucus vesiculosus, max.MW peak 126.7 kD 2. Fucoidan VG49, Fucus vesiculosus, hydrolyzed sampleof 5307002 of lower MW, max. MW peak 22.5 kD 3. Fucoidan VG50,Ascophyllum nodosum, max. MW peak 149.7 kD 4. Fucoidan VG56, Undariapinnatifida, low charge (low sulphation) 5. Fucoidan VG57, Undariapinnatifida, high charge (high sulphation, deacetylated)Fucoidan GFS(5508005), Undaria pinnatifida, depyrogenated 6. Fucoidan GFS(L/UPF-1008), Undaria pinnatifida, hydrolyzed depyrogenated, max. MWpeak 54 kD 7. Fucoidan GFS + Ca (L/UPF-1108), Undaria pinnatifida,hydrolyzed depyrogenated, max. MW peak 32 kD 8. Fucoidan GFS(L/FVF-01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD 9.Fucoidan GFS (L/FVF-01092), Fucus vesiculosus, depyrogenated, max. MWpeak 260 kD 10. Fucoidan GFS (L/FVF-01093), Fucus vesiculosus,hydrolyzed depyrogenated, max. MW peak 36 kD 11. Maritech ® Eckloniaradiata extract 12. Maritech ® Ecklonia maxima extract 13. Maritech ®Alaria esculenta extract 14. Maritech ® Macrocystis pyrifera extract 15.Maritech ® Sargassum fusifome extract 16. Maritech ® Cladosiphon spextract 17. Maritech ® Durivellea potatorum extract 18. Maritech ®Laminiaria digitata extract 19. Maritech ® Fucus polyphenol complexextract 20. Maritech ® Ascophyllum nodosum extract 21. Maritech ® Immunetrial Fucoidan Blend 22. Maritech ® Capsules 23. Depyrogenated Eckloniaradiata 24. Depyrogenated Alaria esculenta 25. Depyrogenated Cladosiphonsp 26. Depyrogenated Sargassum fusiformis 27. Depyrogenated Eckloniamaxima 28. Depyrogenated Macrocystis pyrifera 29. Fucus evanescens 30.Fucus distichus 31. Phyllospora comosa 32. Harmosira banksii 33.Lessonia nigescencs

Example 3 Procoagulant Mechanism of Fucoidans

The following experiments were performed and demonstrate a previouslyunknown procoagulant mechanism of fucoidan. The procoagulant activity ofseveral fucoidans was characterized by calibrated automatedthrombography in tissue factor (TF)-dependent experiments and by usingcoagulation factor-deficient plasmas. Spiking experiments with purifiedcoagulation factors or inhibitory antibodies verified the mechanismidentified. Fucoidan-improved thrombin generation (TG) was TF-dependent.Stimulatory activity was most pronounced without TF. Improvement of TGin FXII-deficient plasma excluded the contact system as a target for theprocoagulant activity. TG experiments without TF using plasmas deficientin proteins of all three coagulation pathways identified FXI as the mostupstream factor responsible for fucoidan-mediated TG. Spiking FXI (30nM) to FXI-deficient plasma restored fucoidan-mediated TG but adding FXIinhibitory antibodies to normal plasma abrogated TG, verifying FXI as atarget for fucoidan. Fucoidan-dependent TG did not improve when FXIa (60μM) was added to FXI-deficient plasma, suggesting FXI activation byfucoidan.

The relevance of this mechanism in hemophilia plasma was studied byaddition of low levels of FVIII (0.2-10%) resulting in a FVIIIconcentration-dependent increase in fucoidan-mediated TG.

As explained above, in vitro characterization of fucoidans suggests thatinhibiting TFPI and accelerating thrombin-dependent FVa formationimproves hemostasis in animal models. These studies describe anotherprocoagulant activity of fucoidans. In particular, FXI activation at lowTF concentrations is a possible mechanism for fucoidan. Identificationof this new mechanism contributes to the understanding of theprocoagulant activities of fucoidans and assists in developing safe andefficient alternatives for treating bleeding disorders.

Example 4 Characterization of Fucoidans

Fucoidans are complex in structure with a high degree of polydispersityand heterogeneity and vary depending on the biological source. A broadrange of analytical tools are applied to understand in depth thebiological activities and structural properties of six differentfucoidan preparations.

The following experiments were performed and demonstrate thatcoagulation properties of NASPs are a function of saccharide content anddegree of sulfation. PPS and fucoidans purified from several brown algae(molecular weight 6-1000 kD) were studied.

Calibrated Automated Thrombography (CAT) Assay

The procoagulant activity was characterized by calibrated automatedthrombography (CAT) in FVIII- and FIX-deficient and FVIII-inhibitedplasma and in combination with hemophilia therapeutics. The mechanism ofthrombin formation as presented herein is shown in FIG. 1.

In the CAT studies, the procoagulant activity of sulfatedpolysaccharides was examined in several plasmas from patients withcongenital coagulation factor deficiencies and FVIII-inhibited normalplasma, in order to study the procoagulant window. Pooled normal plasmaor plasmas from patients with congenital coagulation factor deficiencieswere obtained from George King, Bio-Medical Inc. Kansas USA. Accordingto the supplier, the residual coagulation factor activity for each ofthe coagulation factor deficient plasmas was lower than 1%. As a modelfor antibody mediated FVIII deficiency fresh frozen pooled normal plasma(George King, Bio-Medical Inc., Kansas, USA) was incubated with hightiter heat inactivated anti-human FVIII plasma raised in goat (4490BU/ml; Baxter BioScience, Vienna, Austria) giving rise to 50 BU/mL. Insome experiments, tissue factor pathway inhibitor (TFPI) activity wasblocked in presence or absence of the fucoidan by either a polyclonalgoat anti-human TFPI antibodies (R&D Systems, AF2974, Minneapolis, US)or a monoclonal anti-TFPI antibody directed against the positivelycharged C-terminus of TFPI (Sanquin White Label Products, MW 1848, cloneCLB/TFPI C-terminus, Amsterdam, The Netherlands) at plasma concentrationof 25 nM or 100 nM, respectively. If not indicated otherwise, theplasmas were mixed with corn trypsin inhibitor (CTI) (HematologicTechnologies, Inc., Essex Junction, Vt., USA), providing a finalconcentration of 40 μg/mL, for specific inhibition of factor XIIa.

Test samples were prepared by dissolving quantities of NASPs of interestin Hepes buffered saline and adding human serum albumin (Sigma-AldrichCorporation, St. Louis, Mo., USA) to a concentration of 5 mg/mL.Reference samples were prepared from reference proteins FVIII Immunate®reference standard (Baxter BioScience, Vienna, Austria); Factor eightinhibitor by-passing activity (FEIBA) reference standard (BaxterBioScience, Vienna, Austria); Novo Seven® recombinant activated FVII(Novo Nordisk A/S, Denmark) and purified human plasma FIX (EnzymeResearch Laboratories, South Bend, Ill., USA). A thrombin calibratorcompound was obtained from Thrombinoscope BV, Maastricht, TheNetherlands.

In particular, the influence of each NASP of interest on thrombingeneration was measured in duplicate via calibrated automatedthrombography in a Fluoroskan Ascent® reader (Thermo Labsystems,Helsinki, Finland; filters 390 nm excitation and 460 nm emission)following the slow cleavage of the fluorogenic substrateZ-Gly-Gly-Arg-AMC (Hemker HC., Pathophysiol Haemost Thromb (2003)33:4-15). To each well of a 96 well micro-plate (Immulon 2HB, clearU-bottom; Thermo Electron), 80 μL of pre-warmed (37° C.) plasma wasadded. For triggering thrombin generation by tissue factor, 10 μL of PPPreagent containing an amount of recombinant human tissue factor (rTF)and phospholipid vesicles composed of phosphatidylserine,phosphatidylcholine and phosphatidylethanolamine (48 μM) (ThrombinoscopeBV, Maastricht, The Netherlands) was added. For studying theprocoagulant activity of NASPs, a final TF concentration of 1 pM wasused to provide FVIII and TFPI sensitivity of the test system.Alternatively, a mix of rTF annoying, Siemens Healthcare DiagnosticsInc., Tarrytown, N.Y., USA) and a phospholipid emulsion composed ofphosphatidylcholine, phosphatidylserine and sphingomyelin(Phospholipid-TGT, Rossix, Mölndal, Sweden) was used, which allowed toadjust the TF concentrations from 0 to 20 pM. Just prior to putting theplate into the pre-warmed (37° C.) reader, 10 μL of test or referencesample or calibrator compound was added. Thrombin generation was startedby dispensing 20 μL of FluCa reagent (Thrombinoscope BV, Maastricht, TheNetherlands) containing fluorogenic substrate and Hepes buffered CaCl₂(100 mM) into each well and fluorescence intensity was recorded at 37°C.

The parameters of the resulting thrombin generation curves werecalculated using the Thrombinoscope™ software (Thrombinoscope BV,Maastricht, The Netherlands) and thrombin calibrator to correct forinner filter and substrate consumption effects. With the thrombincalibrator as a reference, the molar concentration of thrombin in thetest wells was calculated by the software. The thrombin amounts at thepeak of each thrombin generation curve (peak thrombin, nM) were plottedagainst the peak thrombin obtained from standard concentrations of areference protein (FVIII Immunate® reference standard, FEIBA referencestandard) and fitted by a non-linear algorithm. Based on thiscalibration, FVIII Immunate®, FEIBA and FIX equivalent activities werecalculated. Other parameters recorded were lag time (time intervalbetween starting measurement and start of thrombin generation), peaktime (time interval between starting measurement and peak thrombin) andendogenous thrombin potential (area under curve of thrombinconcentration versus time).

By CAT, the procoagulant window of sulfated polysaccharides inhemophilic plasma spanned more than two orders of magnitude with maximumeffects being equivalent to (mU/mL) 730-940 FVIII, 32-80 FIX and590-1230 FEIBA. As such, NASPs of interest combined with FVIII, FEIBA,FIX or FVIIa had an additive procoagulant effect.

An example of data acquired by calibrated automated thrombagraphy assayis illustrated in FIG. 2. Results from CAT assays as well as acomparison with hemophilia therapeutics by CAT are summarized in Tables2-4, below. As demonstrated in these results, some NASPs of interestincrease thrombin generation in the absence of CTI at higherconcentrations.

TABLE 2 CAT Evaluation of Compounds (1) Procoagulant Optimal FEIBAwindow Conc Equi activity NASP (μg/mL) (μg/mL) (mU/mL) 5307002  0.1-150*1.9 399 (54-186) VG49  0.2-150* 5.6 369 (70-134) VG50  0.1-150* 1.9 352(20-99)  VG56 16.7-150* 150    162 (58-162) VG57  0.1-50 1.9 357(41-297) 5508005 0.05-100 3.7 460 (35-182) L/UPF-1008 0.14-100 3.7 290(33-242) L/UPF-1108 0.14-100 3.7 296 (36-214) L/FVF-01091 0.05-100 3.7601 (29-298) L/FVF-01092 0.14-100 11.1  476 (56-336) L/FVF-010930.14-300 11.1  441 (32-143) Maritech ® Ecklonia radiata 0.05-100 11.1 657 (51-354) extract Maritech ® Ecklonia maxima 0.14-100 11.1  896(78-629) extract Maritech ® Alaria esculenta 0.14-300* 11.1  375(55-155) extract Maritech ® Macrocystis 0.05-100 1.2 551 (29-58)pyrifera extract Maritech ® Sargassum fusifome 0.41-300* 33.3  474(58-440) extract Maritech ® Clados sp. extract  3.7-300* 300*   173(21-173) Maritech ® Durivellea potatorum 0.41-300 33.3  569 (65-400)extract Maritech ® Laminaria digitata 0.05-33.3 3.7 676 (62-253) extractMaritech ® Fucus polyphenol 0.41-300* 11.1  538 (87-248) complex extractMaritech ® Ascophyllum 0.41-300* 33.3  675 (51-119) nodosum extractMaritech ® Immune trial Fucodian 0.05-100 3.7 551 (25-391) BlendMaritech ® Capsules 100 0.41-300* 11.1  491 (84-372) Maritech ® Capsules200 0.14-300* 3.7 534 (40-429) Depyrogenated Ecklonia radiata 0.05-33.33.7 465 (49-480) Depyrogenated Alaria esculenta 0.14-100 3.7 391(96-291) Depyrogenated Cladosiphon sp. — — — Depyrogenated Sargassum0.14-300* 33.3  416 (22-236) fusiformis Depyrogenated Ecklonia maxima0.14-100 11.1  674 (62-482) Depyrogenated Macrocystis 0.05-33.3 33.3 707 (61-707) pyrifera Fucus evanescens 0.14-100 3.7 523 (70-346) Fucusdistichus 0.14-300* 3.7 422 (49-23) Phyllospora comosa 0.41-300* 11.1 294 (62-145) Hamosira banksii 0.14-300* 33.3  585 (39-308) Lessonianigescencs 0.41-300* 100    429 (43-157)

TABLE 3 CAT Evaluation of Compounds (2) Procoagulant Optimal Thrombinwindow Conc. Peak EC₅₀ NASP (μg/mL) (μg/mL) (%) (μg/mL) BAX513 0.05-1001.23 113.0 0.25 F.v. 5307002 0.41-100 1.23 143.5 0.36 F.v. 53080040.41-100 1.23 135.5 0.46 F.v. 5308005 0.41-100 1.23 138.6 0.40 F.v.VG201094A 0.41-300 11.10 79.8 1.64 F.v. VG201094B 0.41-300 3.70 77.71.42 F.v. VG201095 0.41-300 3.70 89.8 0.95 F.v. VG201097 1.23-300 11.1085.2 2.50 F.v. L/FVF1091 0.05-100 1.23 128.8 0.16 F.v. VG201096A0.14-100 1.23 111.1 0.29 F.v. VG201096B 0.14-100 1.23 110.8 0.23 F.v.DS100110A 0.14-100 1.23 113.8 0.26 F.v. L/FVF1092 0.05-300 1.23 121.00.50 F.v. VG201098A 0.05-300 1.23 131.9 0.54 F.v. VG201098B 0.14-3001.23 121.8 0.47 F.v. L/FVF1093 0.14-300 3.70 116.1 0.82 F.v. DS100111C0.14-300 3.70 147.0 0.52 U.p. 5508005 0.14-100 1.23 107.2 0.36 U.p.5508004 0.14-100 1.23 111.5 0.22 U.p. DPGFS03 0.14-100 1.23 101.2 0.27U.p. UPF200911032 0.41-100 3.7 104.9 1.24 E.m. DS100109C 0.05-100 3.7211.8 1.36 E.m. dep DS100112A 0.05-100 3.7 183.7 0.82 M.p. MPF120080020.14-100 1.23 103.4 0.29 M.p. dep VG201099A 0.14-100 1.23 104.7 0.27M.p. dep VG201099B 0.14-100 1.23 112.0 0.40

TABLE 4 CAT Evaluation of Compounds (3) Advate Equiv. at opt. FEIBAEquiv. at opt. concentration concentration NASP (mU/mL) (mU/mL) F.v.5307002 1430 456 F.v. L/FVF 1091 1540 458 F.v. L/FVF 1092 1080 520 F.v.L/FVF 1093 670 324 U.p. 5508005 1260 445 E.m. DS100112A >2000 833 NormalPlasma 880 322Activated Partial Thromboplastin Time (aPTT) Assay

The anti-coagulant activity was characterized Activated PartialThromboplastin Time (aPTT) Assay. The aPTT assay is performed asdescribed above. Briefly, 50 μL of thawed human plasma (normal orhemophilic) is added to test tubes. 5 μl of saline (e.g. Sigma) or 5 μlof test agent (e.g., NASP) dissolved in saline is mixed with 50 μl ofplasma. aPTT reagent (e.g. STA APTT, Roche) is reconstituted in 5 mldistilled water and 50 μL of the reconstituted solution containing theAPTT reagent is added to each test tube and incubated for 2-3 minutes at37° C. Afterwards 50 μL of 25 mM CaCl₂ is added to initiate clotting.All pipetting steps and plasma clotting time measurements are carriedout with an ACL Elite Pro (Beckman Coulter) instrument. The experimentalsetup and mechanism of aPTT assay as presented herein is shown in FIG.3.

An example of data acquired by activated partial thromboplastin timeassay is illustrated in FIG. 4. Results from aPTT assays as well as theevaluation of pro- and anti-coagulant activity as compared with resultsobtained by CAT are summarized in Table 5, below.

TABLE 5 aPTT Assay: Pro- and Anti-coagulant Activity Evaluation 50%Increase Clotting Time EC₅₀ (μg/mL) (μg/mL) Ratio NASP aPTT CAT aPTT/CATBAX513 7.0 0.3 23.3 F.v. 5307002 7.0 0.4 17.5 F.v. 5308004 6.0 0.5 12.0F.v. 5308005 6.5 0.4 16.3 U.p. 5508005 4.5 0.4 11.3 U.p. 5508004 2.3 0.211.5 U.p. DPGFS03 3.6 0.3 12.0 U.p. UPF200911032 6.55 1.24 5.3 E.m.DS100109C 9.8 1.36 7.2 E.m. DS100112A 8.7 0.8 10.9 E.m. DS100155A 4.31.1 3.9 E.m. DS100155B 5.5 1.0 5.5 E.m. DS100155C 5.0 0.9 5.6 F.v.L/FVF1091 6.5 0.2 32.5 F.v. L/FVF1092 9.9 0.5 19.8 F.v. VG201094A 25.211.64 15.4 F.v. VG201094B 23.36 1.42 16.5 F.v. VG201095 19.41 0.95 20.4F.v. VG201096A 6.3 0.3 21.0 F.v. VG201096B 6.2 0.2 31.0 F.v. VG20109730.09 2.50 12.0 F.v. VG201098A 14.8 0.5 29.6 F.v. VG201098B 16.2 0.532.4 F.v. VG2010100A 5.0 0.6 8.3 F.v. VG2010100B 5.0 0.6 8.3 F.v.VG2010100C 5.3 0.8 6.6 F.v. L/FVF1093 11.9 0.8 14.9 F.v. DS100161A 7.70.5 15.4 F.v. DS100161B 7.8 0.5 15.6 F.v. DS100161C 8.4 n/d n/a F.v.DS100161D 8.2 n/d n/a F.v. DS100161E 6.9 n/d n/a F.v. DS100110A 6.1 0.2623.5 F.v. DS100111C 8.9 0.5 17.8 F.v. DS100159A 6.6 0.5 13.2 F.v.DS100159B 6.5 0.4 16.3 F.v. DS100160A 6.7 0.4 16.8 M.p. MPF12008002 6.340.29 21.9 M.p. dep VG201099A 5.66 0.27 21.0 M.p. dep VG201099B 4.97 0.412.4CAT Assay and aPTT Assay—Activity of Low Molecular Weight Fucoidans

Low molecular weight fucoidans were obtained by fractionation using sizeexclusion chromotagraphy as described in detail in Example 5, below.Size exclusion chromotagraphy was used to obtain fucoidans (Fucusvesiculosus) having a molecular weight which ranged from less than 1 to30 kilodaltons. Fractions obtained from size exclusion chromotagraphyhad molecular weight ranges of: a) less than 1 kilodalton; b) 1 to 30kilodaltons; c) less than 30 kilodaltons; d) 10 to 30 kilodaltons; e) 1to 10 kilodaltons and f) 5 to 30 kilodaltons. Each fraction was studiedby CAT assay and aPTT assay, the method as described in detail above,and compared to the corresponding unfractionated fucoidan sample.Results for the pro- and anticoagulant activity is shown in Table 6,below.

As can be seen from the results, low molecular weight fucoidans possesssimilar activities as compared to unfractionated fucoidan and are atleast as effective in enhancing coagulation.

TABLE 6 Activity of Low Molecular Weight Fucoidans MW Range MW by 50%Defined by SEC- Increase EC₅₀ Ratio filter cutoff MALLS aPTT CAT aPTT/Lot # (kD) (kD) μg/mL μg/mL CAT DS1001104A >1 kD 156 5.6 0.21 26.7DS1001104D 1-30 kD 51 >60 2.92 >20.6 DS1001106A <30 kD 91 6.4 0.20 32.0DS1001106B 10-30 kD 28 10.1 0.30 33.7 DS1001106C 1-10 kD 6.7 43.9 2.4617.8 DS1001108B 5-30 kD 18 15.1 0.45 33.6 F.v. VG201096 B Ø 110 kD 1106.2 0.17 36.5

CAT Assay and aPTT Assay—Activity and SEC chromatography of F.v.L/FVF-1091

Size exclusion chromotagraphy was used to fractionate a sample of F.v.L/FVF-1091 fucoidan. Five fractions were collected and structuralcharacteristics were studied by NMR, ion-exchange chromotagraphy andelemental analysis, the methods described in greater detail in Example5, below. Structural characteristics for the fractions are shown inTable 7, below.

TABLE 7 Structural Characteristics of Fractions from SEC chromatographyof F.v. L/FVF-1091 Fraction # MW (kDa) Sulfur (wt %) Sulfate (wt %) S1436.1 9.3 30.0 S2 135.0 10.3 33.2 S3 54.1 10.0 32.2 S4 30.2 11.1 35.6 S510.6 9.9 31.7

Each fraction was studied by CAT assay and aPTT assay, the method asdescribed in detail above, and compared to the correspondingunfractionated fucoidan sample. Results for the pro- and anticoagulantactivity is shown in Table 8, below.

As can be seen from the results, low molecular weight fractions possessbetter activities as compared to unfractionated fucoidan and are atleast as effective in for enhancing coagulation.

TABLE 8 Activity of Fractions from from SEC chromatography of F.v.L/FVF-1091 50% Increase Clotting Time EC₅₀ (aPTT) (CAT) Ratio Fraction #μg/mL μg/mL aPTT/CAT Starting Material 3.6 0.47 8 S1 6.1 0.29 21 S2 6.10.3 20 S3 7.4 0.35 21 S4 15.7 0.78 20 S5 7.2 0.21 34

Thromboelastography Rotation Thromboelastometry (TEG-ROTEM) Assay

For the TEG studies, blood samples from a healthy individual were drawninto citrated Venoject® tubes (Terumo Europe, Leuven, Belgium (127mmol/L)) mixing one part of citrate with nine parts of blood by a 21-Gbutterfly needle. The first tube aspirated was discarded. A proportionof these blood samples were incubated with high titer heat inactivatedanti-human FVIII antiserum raised in goat (3876 BU/ml; BaxterBioScience, Vienna, Austria) resulting in 51 or 150 BU/mL. Test sampleswere prepared by dissolving quantities of sulfated polysaccharide inHepes buffered saline and adding human serum albumin (Sigma-AldrichCorporation, St. Louis, Mo., USA) to a concentration of 5 mg/mL. Acontrol sample was prepared in which no sulfated polysaccharide wasincluded.

Continuous visco-elastic assessment of human whole blood clot formationand firmness was performed by rotation thromboelastography with wholeblood preparations in the presence or absence of sulfatedpolysaccharides. Briefly, blood was added into a disposable cuvette in aheated cuvette holder. A disposable pin (sensor) was fixed on the tip ofa rotating axis. The axis was guided by a high precision ball bearingsystem and rotates back and forth. The axis was connected with a springfor the measurement of elasticity. The exact position of the axis wasdetected by the reflection of light on a small mirror on the axis. Theloss of elasticity when the sample clots lead to a change in therotation of the axis. The data obtained were analyzed on a computer andvisualized in a thromboelastogram. The thromboelastogram showselasticity (mm) versus time (s). An elasticity of close to zero wasobserved before clot formation begins Mirror image traces above andbelow the zero line indicated the effect of clot formation on therotation of the axis.

Recordings were made using a ROTEM thromboelastography coagulationanalyzer (Pentapharm, Munich, Germany) at 37° C. Before starting eachexperiment, the citrated whole blood was mixed with corn trypsininhibitor (CTI) (Hematologic Technologies, Inc., Essex Junction, Vt.,USA) providing a final concentrations of 37 to 62 μg/mL for specificinhibition of FXIIa, in order to inhibit FXIIa-mediated contactactivation. The analytical set-up was as follows: To 20 μL of testsample or control, 300 μL of pre-warmed (37° C.) CTI treated citratedwhole blood was added, followed by 20 μL of a 1:15 dilution of TF PRPreagent containing recombinant human tissue factor (rTF, 3 pM) (TS40,Thrombinoscope BV, Maastricht, The Netherlands). Coagulation wasinitiated by the addition of 20 μL 200 mM CaCl₂ (star-TEM®, Pentapharm,Munich, Germany) and recordings were allowed to proceed for at least 120min. The final concentration of rTF in the assay was 11 or 44 fM.

The thromboelastographic parameters of clotting time (CT), clotformation time (CFT) and maximum clot firmness (MCF) were recorded inaccordance with the manufacturer's instructions. CT is defined as thetime from the start of measurement to the start of clot formation. CFTis defined as the time from the start of clot formation until anamplitude of 20 mm is reached. MCF is the maximum difference inamplitude between the two traces during the assay. The first derivativeof the data of the thromboelastogram were plotted to obtain a graph ofvelocity (mm/s) against time (s). From this graph, the maximum velocity(maxV) was determined. The time at which the maximum velocity wasobtained (maxV-t) was also determined.

As explained above, the effect of various NASPs on thromboelastographicparameters was tested in FVIII-inhibited blood at two and fourconcentrations, respectively. Two controls were performed in which nofucoidan was present. One used FVIII-inhibited blood and the other usednormal blood. An example of data results from an ROTEMthromboelastography coagulation analyzer is shown in FIG. 5.

Results from data obtained from a first ROTEM thromboelastographycoagulation analyzer are summarized in Table 9. The FVIII-inhibitedblood had a characteristically long clotting time and clot formationtime. The clotting time and clot formation time were both shorter in theFVIII-inhibited blood containing fucoidan, with the fucoidan exerting aconcentration dependent effect on both parameters. Fucoidan also reducedCT and CFT in normal blood.

Data from a second set of experiments using ROTEM thromboelastographycoagulation analyzer such as those illustrated in FIG. 6 are summarizedin Table 10. As demonstrated by the data presented in Table 10, FucoidanSmart City and fucoidan derived from Laminaria japonica enhancescoagulation parameters of FVIII inhibited blood and normal blood.

TABLE 9 Thromboelastography Rotation Thromboelastometry (TEG-ROTEM)Assay (1) Clotting parameters MCF Activity Fucoidan/Type of Blood CT (s)CFT (s) (mm) (%) Control - FVIII-inhibited blood 2447 881 55 — Undariapinnatifida 10 nM - FVIII 1163 419 55 — inhibited blood Undariapinnatifida 100 nM - FVIII 956 330 50 — inhibited blood Control - Normalblood 869 274 45 — Undaria pinnatifida 10 nM - Normal 767 225 46 — bloodUndaria pinnatifida 100 nM - 382 105 54 — Normal blood Control -FVIII-inhibited blood 4829 2054 — 0 Laminaria japonica 0.4 μg/mL - 29171431 — 68 FVIII inhibited blood Laminaria japonica 1.2 μg/mL - 2069 79863 98 FVIII inhibited blood Laminaria japonica 3.7 μg/mL - 1656 525 63.5113 FVIII inhibited blood Laminaria japonica 11.1 μg/mL - 1650 421 60113 FVIII inhibited blood Control - Normal blood 1557 373 52 100Laminaria japonica 0.4 μg/mL - 1458 271 55 — Normal blood Laminariajaponica 1.2 μg/mL - 1121 244 54 — Normal blood Laminaria japonica 3.7μg/mL - 651 224 52 — Normal blood Laminaria japonica 11.1 μg/mL - 744317 52 — Normal blood Control - FVIII-inhibited blood 3474 1978 — 0 F.vesiculosus L/FVF1091 1970 1074 58 72 0.4 μg/mL - FVIII inhibited bloodF. vesiculosus L/FVF1091 1417 606 61 99 1.2 μg/mL - FVIII inhibitedblood F. vesiculosus L/FVF1091 1299 421 60.5 104 3.7 μg/mL - FVIIIinhibited blood F. vesiculosus L/FVF1091 1450 418 60.5 97 11.1 μg/mL -FVIII inhibited blood Control - Normal blood 1390 313 50.5 100 Control -FVIII-inhibited blood 3789 2073 — 0 F. vesiculosus 5307002 2284 1302 —79 0.4 μg/mL - FVIII inhibited blood F. vesiculosus 5307002 1729 81161.5 109 1.2 μg/mL - FVIII inhibited blood F. vesiculosus 5307002 1364484 62 128 3.7 μg/mL - FVIII inhibited blood F. vesiculosus 5307002 1530357 60 119 11.1 μg/mL - FVIII inhibited blood Control - Normal blood1892 388 50 100 Control - FVIII-inhibited blood 4150 1820 — 0 F.vesiculosus L/FVF1092 2363 1125 — 72 0.4 μg/mL - FVIII inhibited bloodF. vesiculosus L/FVF1092 1587 662 61.5 103 1.2 μg/mL - FVIII inhibitedblood F. vesiculosus L/FVF1092 1428 464 62 109 3.7 μg/mL - FVIIIinhibited blood F. vesiculosus L/FVF1092 1184 240 60 119 11.1 μg/mL -FVIII inhibited blood Control - Normal blood 1659 358 51 100 Control -FVIII-inhibited blood 3262 2103 — 0 F. vesiculosus L/FVF1093 2362 1054 —51 0.4 μg/mL - FVIII inhibited blood F. vesiculosus L/FVF1093 1970 1015— 74 1.2 μg/mL - FVIII inhibited blood F. vesiculosus L/FVF1093 1669 82860 91 3.7 μg/mL - FVIII inhibited blood F. vesiculosus L/FVF1093 1316459 60 111 11.1 μg/mL - FVIII inhibited blood Control - Normal blood1510 291 53 100 Control - FVIII-inhibited blood 4339 — — 0 Undariapinnatifida 5508005 2891 1362 — 52 0.4 μg/mL - FVIII inhibited bloodUndaria pinnatifida 5508005 1534 524 62.5 101 1.2 μg/mL - FVIIIinhibited blood Undaria pinnatifida 5508005 1215 284 62 113 3.7 μg/mL -FVIII inhibited blood Undaria pinnatifida 5508005 1197 272 57 114 11.1μg/mL - FVIII inhibited blood Control - Normal blood 1574 343 52 100Control - FVIII-inhibited blood 3943 — — 0 Ecklonia maxima DS100112A2334 1192 — 80 0.4 μg/mL - FVIII inhibited blood Ecklonia maximaDS100112A 1285 338 66 132 1.2 μg/mL - FVIII inhibited blood Eckloniamaxima DS100112A 833 141 63 154 3.7 μg/mL - FVIII inhibited bloodEcklonia maxima DS100112A 919 160 59 150 11.1 μg/mL - FVIII inhibitedblood Control - Normal blood 1926 398 56.5 100

TABLE 10 Thromboelastography Rotation Thromboelastometry (TEG-ROTEM)Assay (2) Clotting parameters Fucoidan/Type of Blood CT (s) CFT (s) MCF(mm) Fucoidan Smart City in Normal Blood Hem A Blood 5033 2025 — SmartCity 2 μg/ml 3061 1102 — Smart City 10 μg/ml 2074 1039 57 Human Blood931 255 52 Fucoidan Smart City in FVIII Inhibited Blood Hem A Blood 45942894 27 Smart City 2 μg/ml 2493 888 55 Smart City 10 μg/ml 1651 613 51Human Blood 1104 283 45 Fucoidan Laminaria japonica in FVIII InhibitedBlood FVIII i. blood 3367 2275 — HemA + Laminaria japonica 2313 1018 53Normal Blood 1374 346 43 NB + Laminaria japonica 939 432 46

Example 5 Structural Characterization of Fucoidan

Fucoidan preparations were characterized based on various structuralcharacteristics.

Degree of Sulfation

The degree of sulfation was determined by elemental analysis using a PE2400 CHN Analyzer and sulfur content was determined by colorimetric atitration. Sulfur content was also verified using inductively coupledplasma mass spectrometry. The results of analysis are summarized inTable 11.

TABLE 11 Degree of Sulfation Sulfur Sulfate - SO₃ (Colorimetric)(Derived) NASP w % w % L. japonica BAX513 5.8 14.5 E.m. DS100112A 6.015.0 E.m. DS100155A 6.7 21.6 E.m. DS100155B 6.7 21.6 E.m. DS100155C 6.119.6 E.m. VG23 6.6 16.5 U.p. 5508005 10.0 25.0 U.p. 5508004 10.0 25.0U.p. DPGFS03 10.0 25.0 U.p. VG56 5.3 16.0 U.p. VG57 10.6 32.1 F.v.5307002 8.7 21.8 F.v. 5308004 9.5 23.4 F.v. 5308005 8.4 21.0 F.v. L/FVF1091 8.7 21.8 F.v. L/FVF1092 7.7 19.3 F.v. L/FVF1093 6.6 16.5 F.v. VG498.6 26 F.v. VG50 8.6 26 F.v. VG2010100A 8.6 27.7 F.v. VG2010100B 9.129.2 F.v. VG2010100C 9.6 31.0 F.v. VG201096A 9.1 22.8 F.v. VG201096B 9.924.8 F.v. VG201098A 5.7 14.3 F.v. VG201098B 5.2 13.0 F.v. DS100111C 6.716.8 F.v. DS100159A 6.8 21.9 F.v. DS100159B 7.3 23.4 F.v. DS100160A 7.223.2

Monosaccharide Content

The monosaccharide content of various NASPs was analyzed by ionchromatography and by nuclear magnetic resonance spectroscopy.

Ion Chromatography

Monosaccharide compositions of various NASPs were analyzed using ionchromatography. A Dionex ICS 3000 system was used to analyze thehydrolysates coupled with a PAD detector. Seven neutral sugars wereapplied in this method as standards. They were Fucose, Rhamnose,Arabinose, Galactose, Glucose, Xylose and Mannose. An example of achromatogram to determine monosaccharide composition is illustrated inFIG. 7. Monosaccharide contents as determined by ion-exchangechromotagraphy of several NASPs of interest are depicted in FIGS. 8-10and summarized in Table 12, below.

IC Condition:

-   Column: Dionex guard column CarboPac® PA10, 2×50 mm, and-   Dionex analytical column CarboPac® PA1, 4×250 mm.-   Mobile phase: 2 mM NaOH-   Flow rate: 1 mL/min-   Column Temp.: 35° C.-   Running time: 30 min

TABLE 12 Fucose Content by Ion-exchange Chromatography NASP % Fucose L.japonica BAX513 39 F.v. 5307002 65 F.v. L/FVF 1091 73 F.v. L/FVF1092 46F.v. L/FVF1093 53 U.p. 5308005 58 E.m. DS100112A 38

Nuclear Magnetic Resonance Spectroscopy

A Bruker Avance III NMR spectrometer with a dual ¹H/¹³C-Cryoprobe wasused to analyze the fucoidan starting material and its fractions. Eachsample was dissolved in ˜0.6 mL D₂O. Qualitative NMR experiments wereused to characterize their structures. One-dimensional ¹H NMR spectrawere obtained using 16 scans, a 90° pulse, a relaxation delay of 20seconds, 32K Data points, and a 2 second acquisition time. The phasesensitive multiplicity edited Heteronuclear Single Quantum Correlation(HSQC), magnitude mode Heteronuclear Multiple Bond Correlation (HMBC)and correlation spectroscopy (COSY) spectra were obtained using 1024data points in the observe domain and 128 points in the seconddimension. Quantitative one-dimensional ¹³C NMR spectra were obtainedusing 3 k scans, a relaxation delay of 5 seconds. Based on the ¹³C NMRspectra of fucoidan starting material and its fractions, their alginateand fucose contents could be calculated. Based on the degree ofcomplexity of the anomeric and fingerprint ranges in ¹³C-NMR, theirheterogeneity order could be roughly ranked on a scale from 1-7, 1 beingthe highest and 7 being the lowest. For example, a ranking of 1indicates a high heterogeneity sample whereas a 7 indicates a lowheterogeneity sample.

-   -   Alginate Content (C %^(alginate) is the % alginate of the total        saccharides, and can be calculated from the fact that:        -   Carbonyl groups are only present in alginate, where there is            one per saccharide.        -   Each sugar residue, from alginate or fucoidan, has one            anomeric carbon per saccharide ring.        -   Therefore:

$\begin{matrix}{{C\mspace{14mu} \%^{alginate}} = {\frac{\int{carbonyls}}{\int{anomerics}} \times 100\%}} & {{Eq}.\mspace{14mu} \lbrack 1\rbrack}\end{matrix}$

-   -   -   where ∫carbonyls=integral of carbonyl groups;            ∫anomerics=integral of anomeric region.        -   The alginate content was calculated and listed in Table 2.            Because the value for F.V. VG preparations was so low, they            were listed as having less than 10% alginate.

    -   Fucose Content (C %^(flucose)) is the % fucose of the neutral        saccharides (total saccharides−alginate), and is based on the        fact that there is one methyl group per fucose residue:

$\begin{matrix}{{C\mspace{14mu} \%^{fucose}} = {\frac{\int{methyls}}{{\int{anomerics}} - {\int{carbonyls}}} \times 100\%}} & {{Eq}.\mspace{14mu} \lbrack 2\rbrack}\end{matrix}$

-   -   -   where f methyls=integral of methyl groups.        -   Equation 2 was used for E.M. DS and F.V. DS preparations            where the alginate content is substantial. The F.V. VG            samples had negligible alginate content, and equation 2 was            simplified to:

$\begin{matrix}{{C\mspace{14mu} \%^{fucose}} = {\frac{\int{methyls}}{\int{anomerics}} \times 100\%}} & {{Eq}.\mspace{14mu} \lbrack 3\rbrack}\end{matrix}$

Example spectra used to determine monosaccharide composition areillustrated in FIG. 11. The monosaccharide content and heterogeneity ofseveral NASPs of interest as determined by NMR are summarized in Table13.

TABLE 13 Monosaccharide Content by Nuclear Magnetic ResonanceSpectroscopy Heterogeneity Order (Scale 1-7, NASP % Fucose % Alginate 1= highest) E.m. DS100112A 46 31 2 E.m. DS100155A 55 32 2 E.m. DS100155B51 27 2 E.m. DS100155C 51 25 2 F.v. L/FVF 1091 91 ≦10 7 F.v. VG2010100A93 ≦10 7 F.v. VG2010100B 89 ≦10 7 F.v. VG2010100C 95 ≦10 7 F.v.DS100111C 52 Low signal 5 F.v. DS100159A 78 12 5 F.v. DS100159B 79 14 5F.v. DS100160A 75 11 5 L. japonica BAX513 40 −24 −1 F.v. 5307002 87 −≦10−7 F.v. L/FVF1092 52 −≦10 −6 F.v. L/FVF1093 54 Low signal −6 U.p.5508005 50 ≦10 −3 *Based on degree of the complexity of anomerics andthe fingerprint region in ¹³C-NMR. The heterogeneity order was roughlyranked from 1 to 7, where the heterogeneity of Bax513 is 1, the highestheterogeneity. Bax513 is not listed in Table 10, but is observed in FIG.11

Molecular Weight Distribution

The molecular weight distribution of various NASPs (e.g., fucoidans) wasanalyzed by size exclusion chromatography, anion exchange fractionation,gel electrophoresis.

SEC Chromatography

Size exclusion chromatography (SEC) was conducted as follows. A 10 mg/mLsample solution was prepared and filtered through a 0.45 μm Ultrafree-MCHV Centrifugal Filter, followed by injection of 200 μl into HPLC. AnAgilent 1100 HPLC system coupled with Wyatt Technology DAWN HELEOS,Quasi-Elastic Light Scattering (QELS), multi-angle laser light scatter(MALLS) and Optilab rEX differential refractive index (dRI) detectorsand a GE Healthcare column, Superdex 200 column, were used tofractionate starting material solution by size. Based on the profile ofRI detector, the NASP (e.g., fucoidan) was fractionated by differentrange of retention time. The molecular weights of fractions weredetermined by analyzing them with same method with MALLS detector.

LC Conditions: Analytical Column: GE Healthcare Superdex 200, 10/300GLMobile Phase: 10% PBS Buffer, pH 7.4

Flow Rate: 1.0 mL/MinuteColumn Temp. AmbientSample Temp. Set at 5° C.

Injection Volume: 100 μL

In one example, where two major peaks appeared in a chromatogram of afucoidan sample, the calculation of the molecular weight of the twopeaks is described below.

Molecular weight calculation Ret. Time (min) V_(e) (mL) K_(av) Ig MW(kDa) MW (Da) Fraction I 19.843 7.9372 0.015706 5.119969 >200,000Fraction II 22.121 8.8484 0.106975 4.961954 90000 V_(e) = volume ofeluent collected V_(o) = column void volume V_(t) = total bed volumeK_(av) = V_(e) − V_(o)/V_(t) − V_(o) Ig MW = Logarithm of Molecularweights

Example molecular weight profiles of some NASPs (e.g., fucoidans) ofinterest are summarized in Table 14, below.

TABLE 14 Molecular Weight Distribution Molecular weight profiles of somefucoidans of interest. Molecular weights are relative to dextran. Maxpeak MW % MW % MW % MW % MW % MW % MW % Sample MW (kDa) >1600k1100-1600k 200-1100k 60-200k 20-60k 5-20k <5k U.p. VG57 — 10.5 4 30 2815 3 16.4 U.p.VG56 — 10.5 2 16 24 26 10.5 12.7 F.v. 5307002 126.7 7 2 1921 15 10 11.9 F.v. VG49 22.5 1 0.5 5 14 30 28 12.0 A.n. VG50 149.7 22 524 18 12 7.5 27.6 U.p. 54 3.1 1.6 16.4 26.0 24.3 12.2 16.4 L/UPF-1008U.p. 32 5.1 0.9 10.7 22.3 30.5 17.9 12.7 L/UPF-1108 F.v. 125 2.1 2.733.5 28.9 14.6 6.2 11.9 L/FVF- 01091 F.v. 260 19.9 6.9 32.7 15.8 7.7 5.012.0 L/FVF- 01092 F.v. 36 0.8 0.5 9.2 20.6 22.7 18.5 27.6 L/FVF- 01093

Anion Exchange Chromatography

Anion exchange chromatography was conducted using a weak anion exchangeGE Healthcare LC system, ÄKTA Purifier 100 system and a DEAE Sepharosefast flow (FF) column (5×22 cm, column volume=431 mL) as follows.

Anion Exchange Chromatography by DEAE FF Column

Two hundred μL of a 10 mg/mL solution of fucoidan sample F.v. V201096Bin 20 mM ammonium acetate pH 8.0 was prepared, filtered through 0.45 μmUltrafree-MC HV centrifugal filter and injected onto DEAE FF column (5mL). Analytes were detected by Phenol-Sulfuric Acid Assay offline.Separation was effected by a salt gradient using the system shown below.

LC conditions:

Mobile Phase: Solvent A, Milli-Q Water; Solvent B, 2 M NaCl

Flow rate: 49 mL/min

Column Temperature Room Temperature

Injection volume: 1.5 mL, 80 mg/mL

Gradient: 0% B, 1 CV; 0-100% B, 16 CV

Collection: 49 mL/tube

Detection: Phenol-sulfuric acid assay offline

Phenol-Sulfuric Acid Assay

The odd-numbered tubes were tested by phenol-sulfuric acid assay. Thisassay was modified from an original method developed by Dubois, et al(Analytical Chemistry, 28, 1956, 350-356), the method of which is,herein incorporated by reference. To 300 μL sample, 100 μL 5% (w/v)aqueous phenol and subsequent 1 mL concentrated sulfuric acid wereadded. The reactions were done by incubation at 100° C. in an oven for10 minutes. After the samples were cooled down to room temperature, theywere transferred to 96 well plate (200 μL) and absorbance was measuredat 490 mm. The chromatograms were generated by these phenol-sulfuricacid assay data.

Agarose Gel Analysis

Fucoidan starting material and lower molecular weight fractions wereanalyzed by agarose gel electrophoresis. The purities and chargeproperties of these highly disperse sulfated polysaccharides wereanalyzed using this method. A Bio-Rad Mini-Sub cell was used to cast thegel. Samples (10-20 μg of each) were applied to a 0.5% agarose gel in0.04 M barium acetate and run for 2 h at 100 mA in 0.05 M1,3-diaminopropane-acetate (pH 9.0). The gel was dyed in 0.2% (w/v)Alcian blue and 2% (v/v) acetic acid aqueous solution for 30 minutes anddestained in Milli-Q water for overnight to clean the background.

PAGE Analysis

Fucoidan starting material and lower molecular weight fractions werealso analyzed by polyacrylamide gel electrophoresis with a Bio-Radmini-gel electrophoresis system. The molecular size properties of thesehighly disperse sulfated polysaccharides were analyzed using thismethod. Each sample (5-10 μg) was combined with one volume of 50% (w/v)sucrose, and the mixture was loaded into a stacking gel of 5% (totalacrylamide) and analyzed with a 15% resolving gel. The upper chamberbuffer composed of 0.2 M Tris and 1.25 M glycine at pH 8.3. The lowerchamber buffer contained 0.1 M boric acid, 0.1 M Tris and 0.01 Mdisodium ethylene diamine tetra-acetic acid (EDTA) at pH 8.3. Resolvinggel contained 13.6% acrylamide and 1.4% N,N′-methylenebisacrylamide and15% sucrose and dissolved in lower chamber buffer. Electrophoresis wasperformed at 150 V for 80-90 min. The gel was dyed with Alcain blue in2% (v/v) acetic acid.

Hydrolysis and Thin Layer Chromatography (TLC) Monitoring

All blanks (1N methanolic HCl), standard mix (2 mg/mL in 1N methanolicHCl) and samples (2 mg/mL in 1N methanolic HCl) were heated forapproximately 24 hours in an 80° C. heating block. The hydrolyzedsolutions were then evaporated to dryness under vacuum at 45-55° C. andreconstituted in water.

The hydrolysates were analyzed by TLC to monitor the completeness ofhydrolysis. The following materials were used to perform the test:

-   -   1. HPTLC silica gel 60 from Merck, Germany    -   2. Developing solvent, 1-Propanol:H₂O=8:3 or Formic acid:        1-butanol: H₂O=6:4:1.    -   3. Stain solvent, diphenylamine-aniline-phosphoric acid reagent        −1 ml of 37.5% HCl, 2 ml of aniline, 10 ml of 85% H₃PO₄, 100 ml        of ethyl acetate, and 2 g of diphenylamine

The samples (˜1.5 μL volumes) were separately loaded onto a TLC plate(˜4×5 cm) and developed with the solvent system. The developed plate wasdried by a heat plate and stained by dipping indiphenylamine-aniline-phosphoric acid reagent for 2 seconds, followed byheating in a 150° C. oven for approximately 10 minutes.

Elemental Analysis

The PE 2400 CHN Analyzer was used for C, H and N measurements. Sulfurwas analyzed by colorimetric titration. These analyses were conducted byIntertek USA, Inc. QTI laboratory.

Lot-to-lot Variability

The lot-to-lot variability of the NASPs of interest were tested using aPhenol-sulfuric acid depolymerization assay as well as a Toluidine BlueAssay.

Phenol-Sulfuric Acid and Toluidine Blue Assays

The quantitation of carbohydrates in NASPs were tested byphenol-sulfuric acid assay. As fucose is a component of fucoidan, it wasused as a standard to help in quantifying the monosaccharide content offucoidans. Phenol-sulfuric acid assay was performed on both knownamounts of fucose and test samples under the sample conditions. Based onthe standard curve generated with various amounts of fucose,carbohydrate content was determined.

Toluidine Blue Assays were performed by conventional means by adding anamount of toluidine blue to fractionated and unfractionated NASPs.Toluidine blue is a cationic dye that binds to sulfates, phosphates andcarboxylates. Different NASPs will show different bindingcharacteristics depending on sulfate and uronic acid content.

Based on the Phenol-Sulfuric Acid and Toluidine Blude Assays, there waslow lot-to-lot variability between samples of NASPs tested, as describedabove.

Example 6 Bioavailability

The bioavailability of fucoidans of interest were studied using CaCo2cell model screening. This method utilizes a human colon carcinoma cellline that expresses a wide range of transporter proteins on its cellmembranes. Cell layers are grown on a membrane surface that separatestwo compartments (24-well plate). An example of the experimental setupfor these experiments is illustrated in FIG. 12. Selected fucoidansamples were dissolved in RPMI cell medium at a concentration of 1 mg/mLand applied onto the cells in the apical compartment. Cells wereincubated at 37° C. in 5% CO₂. Medium samples were removed from thebasolateral and apical compartment at different time points. Thecondition of the cell layer was monitored by measurement of thetransepithelial electrical resistance (TEER). Samples were analyzed bythrombin generation assay (CAT), as described above in FVIII inhibitedhuman plasma. NASP concentration was calculated based on activity fromCAT assay. All fucoidan samples were diluted in such a way that thesample concentration was in the range of increasing procoagulantactivity. Based on the initial load concentration values, apical andbasolateral concentrations were determined at 2 hour increments (e.g., 2hours, 4 hours, 6 hours, 8 hours, including 24 hours). Based on thedetermined basolateral concentrations, the percent resorption wasdetermined for each compound. An example of cell resorption of thefucoidan Fucus vesiculosus, L/FVF-1091 as a function of time asdetermined by the CaCo2 system is illustrated in FIG. 13. The results ofbioavailability studies using the CaCo2 cell model screening of NASPs ofinterest as described herein are summarized below in Table 15, below.

TABLE 15 Bioavailability % Resorption Range % Resorption Range Fucoidan(2-8 hours) (24 hours) F.v. L/FVF 1091 - Set 1 0.2-2.8 2.0-5.7 F.v.L/FVF 1091 - Set 2   0-0.3   0-0.9 F.v. L/FVF 1091 - Set 3   0-0.60.2-1.3 F.v. VG 49 0.6-0.7 0.6-0.7 F.v. L/FVF 1092 0.7-1.5 1.5-1.8 F.v.5307002 - Set 1 0.4-0.9 1.2-3.4 F.v. 5307002 - Set 2   0-1.3 0.6-3.7F.v. 5307002 - Set 3 0.7-1.0 0.7-1.0 U.p. 5508005 - Set 1 0.2-6.1 0.6-18.4 U.p. 5508005 - Set 2 0.5-2.0 2.0-7.0 U.p. 5508005 - Set 30.3-3   2.0-23.0 U.p. 5508005 - Set 4   0-1.5 0.4-5.0 F.v. L/FVF 1093 -Set 1  0.4-12.1 15.2-47.6 F.v. L/FVF 1093 - Set 2 0.2-0.7 0.4-0.6 F.v.L/FVF 1093 - Set 3   0-0.5  1.4-21.5 E.m. DS100112A - Set 1  0.2-10.9 4.4-16.3 E.m. DS100112A - Set 2   0-0.4 0.3-0.4 E.m. DS100112A - Set 30   28.4-63.5 L. japonica BAX513 - Set 1 0.5-1.7 2.0-4.7 L. japonicaBAX513 - Set 2 0.4-3.9  7.0-10.3 L. japonica BAX513 - Set 3 0.2-0.60.5-2.6 L. japonica BAX513 - Set 4   0-0.3   0-0.6 L. japonica BAX513 -Set 4 0.2  7.9-14.8

Example 7 Effect of Fucoidan on TFPI Function

The effect of fucoidan on TFPI function was tested using the variousfucoidan compositions. In particular, the effect of fucoidan on thefunction of TFPI was tested by calibrated automated thrombography at lowTF concentration (1 μM) in pooled normal plasma in the presence andabsence of antibodies which inhibit the activities of TFPI. Controlswere performed in which no fucoidan was present. Results from studies onthe mode of action on TFPI with various fucoidans in TFPI depleted andFVIII inhibited plasma or normal plasma are shown in Tables 16-17,below. An example of CAT assay results is shown in FIG. 14.

Results from studies on the mode of action on TFPI with some fucoidansof interest in normal plasma are shown in Table 18, below. An example ofCAT assay results from these studies is shown in FIG. 15.

Results from studies on the mode of inhibition of TFPI with somefucoidans of interest in FVIII-inhibited dFX Plasma are shown in Table19 below. An example of CAT assay results from these studies is shown inFIG. 16.

As can be seen from the results presented herein, fucoidans of theinvention increased peak thrombin of pooled normal plasma and shortenedthe time to peak thrombin when added at optimal concentration which isconsistent with its procoagulant activity. By blocking the activities ofTFPI with a polyclonal anti-TFPI antibody, fucoidan parameters ofthrombin generation did not change which indicated that fucoidaninterferes with the function of TFPI.

To further explore the functional site on TFPI which is targeted byfucoidan, a monoclonal antibody directed against the basic C-terminus ofTFPI was used. Addition of the antibody improved thrombin generation byincreasing peak thrombin to 151 nM and reducing peak time to 7.8 min.When Laminaria japonica fucoidan was added to such a test system, it didnot change parameters of thrombin generation. This indicates thatfucoidan interferes with the C-terminus of TFPI, which is known to be offunctional importance.

To further show that Laminaria japonica fucoidan interacts with theC-terminus of TFPI, recombinant C-terminally truncated TFPI (TFPI 1-160)was added to a test system in which the activity of full-length TFPI wasblocked by an antibody directed against the C-terminus of TFPI. Additionof TFPI 1-160 reduced peak thrombin from 151 nM to 57 nM and increasedpeak time from 7.8 min to 11.0 min. Addition of Laminaria japonicafucoidan to this system did not change the parameters of thrombingeneration. This confirms that Laminaria japonica fucoidan interacts andinterferes with the activity of C-terminal TFPI regions. Additionalstudies were conducted using fucoidans of interest as described aboveand gave analogous results. As such, fucoidans or interest may beemployed to inhibit activity by TFPI in FVIII inhibited plasma.

TABLE 16 Fucoidan Laminaria japonica/TFPI Mode of Action Peak thrombinPeak time Fucoidan/protein (nM) (min) Control 83 11.3 Laminaria japonicafucoidan 1.2 μg/mL 171 8.2 Control - polyclonal anti TFPI 250 6.5Laminaria japonica fucoidan 1.2 μg/mL - 247 6.7 polyclonal anti TFPIControl - anti TFPI C-terminus 151 7.8 Laminaria japonica fucoidan 1.2μg/mL - 149 8.0 anti TFPI C-terminus Control - anti TFPI C-terminus +TFPI 1-160 57 11.0 Laminaria japonica fucoidan 1.2 μ/mL - anti 59 11.3TFPI C-terminus + TFPI 1-160

TABLE 17 Fucoidans/Inhibition of TFPI - in ΔTPFI FVIII inhibited PlasmanM Peak Thrombin TFPI160 + Fuc. hflTFPI + Fuc. Fuc. Buffer - No Fucoidan20.5 17.9 86.23 Laminaria japonica 20.74 83.9 79.35 F.v. 5307002 20.3287.93 76.72 F.v. L/FVF1091 21.13 80.06 76.13 F.v. L/FVF 1092 21.11 89.5378.12 F.v. L/FVF 1093 19.82 89.18 79.9 F.v. 5508005 19.71 88.6 79.95E.m. DS100112A 37.86 104.24 96.32

TABLE 18 Fucoidans/Inhibition of TFPI - in Normal Plasma dTFPI- dTFPINP + NP + C-Term + dTFPI C-Term + nM Peak Thrombin Fuc Fuc Fuc TFPI160 +Fuc Buffer - No Fucoidan 57.02 216.6 162.59 75.2 Laminaria japonica139.52 210.85 148.99 71.13 F.v. 5307002 138.01 213 153.81 74.66 F.v.L/FVF1091 129.39 210.74 155.91 73.75 F.v. L/FVF 1092 142.44 192.07134.93 72.03 F.v. L/FVF 1093 138.84 205.23 140.87 71.49 F.v. 5508005134.81 201.38 135.75 69.38 E.m. DS100112A 213.29 224.45 181.84 164.14

TABLE 19 Fucoidans/Inhibition of TFPI - in FVIII inhibited dFX PlasmadFX + dFX/ dFX + Fuc dTFPI + Fuc dFX/dTFPI + nM Peak Thrombin (EC50) Fuc(EC50) (EC90) Fuc (EC90) Buffer - No Fucoidan 27.15 66.28 27.15 66.28Laminaria japonica 33.37 65.72 51.38 69.3 F.v. 5307002 39.43 60.88 46.8463.9 F.v. L/FVF1091 36.06 67.22 51.64 69.27 F.v. L/FVF 1092 37.57 71.9457.94 79.19 F.v. L/FVF 1093 38.95 73.85 59.18 81.54 F.v. 5508005 49.6100.01 75.22 96.29 E.m. DS100112A 46.44 91.04 82.4 87.68

Surface plasmon resonance experiments (Biacore 3000, G.E. Healthcare)were also used to study the interaction of fucoidan with human TFPIproteins. The proteins used were full-length TFPI (aa 1-276) andC-terminally truncated TFPI (aa 1-160). The C-terminally truncated TFPI1-160 lacks the negatively charged C-terminus and the Kunitz domain 3Full-length TFPI (flTFPI) was constitutively expressed by SKHep1 cellsand purified by a multistep purification protocol using conventionalpurification devices and columns TFPI 1-160 was expressed by E. coli ininclusion bodies and was refolded and purified by a multisteppurification protocol using conventional purification devices andcolumns. The proteins were covalently coupled to a CM5 chip (GEHealthcare) using conventional amine coupling chemistry at pH 4.5 (10 mMNaAcetate) resulting in immobilization of 900 RU for flTFPI and 500 RUfor TFPI 1-160, respectively.

For the binding assays the surfaces were equilibrated at a flow rate of30 μL/min with HBS-P buffer (0.01M Hepes pH 7.4; 0.15M NaCl; 0.005%Surfactant P20) (GE Healthcare) to which 1% Tween 80 (Merck) was added.After 75 seconds, the Laminaria japonica fucoidan dissolved in HBS-P, 1%Tween 80 was injected for 450 seconds at concentrations ranging from0.02 μg/mL to 250 μg/mL followed by a dissociation time of 475 seconds.The chip was regenerated by injecting 10 μL of 2.5 M NaCl followed by 10mM NaOH, 1 M NaCl. HBS-P buffer plus 1% Tween 80 was used throughout thebinding assays. Each sensorgram was referenced against buffer and theblank cell, respectively.

Results are shown in FIG. 17. Fucoidan reacted with flTFPI in aconcentration dependent manner, whereas no binding was observed withC-terminally truncated TFPI 1-160. This indicates that fucoidan (e.g.,Laminaria japonica) binds in the C-terminal region of TFPI, which isknown to be of functional importance.

Example 8 In Vitro Studies in Animal Plasma

Studies of fucoidans of interest were conducted in animal plasma inorder to identify one or more animal species for future in vivo studiesin which fucoidan response can be observed. CAT assays were conducted bytitration of fucoidan in a wide concentration range (0.02-300 μg/mL) innormal and if possible, FVIII-inhibited animal plasma Animal plasmasfrom human, cynomolgus monkey, guinea pig, rat, dog, rabbit, mouse andminipig were tested. CAT conditions were optimized for each animalspecies, in accordance with the concentration of FVIII inhibitor,concentration of tissue factor and plasma dilution. Assay conditions foreach animal species are shown in Table 20, below. Coagulant effects weremeasured in a therapeutic window of up to about 300 μg/mL.

Based on animal plasma studies, cynomolgus monkey, guinea pig and ratwere determined to be suitable candidates for possible in vivo studiesto determine fucoidan response. Plasma from dog, rabbit, mouse andminipig were determined to be less suitable candidates for in vivostudies.

In accordance with in vitro animal plasma test studies, two guinea pigmodels were developed to evaluate in vivo activity of fucoidans ofinterest: a carotid occlusion model and ex-vivo TEG analysis of wholeblood.

TABLE 20 CAT Assay Conditions for in vitro studies in Animal PlasmaFVIII Inhibitor Concentration TF Concentration Species Plasma Dilution(BU/mL) (pM) Human 1:1.5  50 1 Rat 1:3   n/a 0.6 Monkey 1:1.5 150 0.6Guinea Pig 1:3   150 0.6 Minipig 1:1.5 300 0.1 Mouse 1:3   150 0.6

Example 9 In Vivo Studies in Guinea Pies

Studies to evaluate the activity of fucoidans ex vivo were conductedusing guinea pigs as animal models. To inhibit endogenous FVIII in theguinea pig an FVIII inhibitor (Z994; 42 BU/kg) was administeredintravenuously 45 minutes prior to blood sampling. Fucoidan preparationF.v. VG201096B at 0.1, 0.4 or 1.6 mg/kg was administered intravenuously5 minutes prior to blood sampling. Puncture of the vena cava caudaliswas performed to sample whole blood. Measurements by thromboelastographywere conducted immediately after sampling the citrated whole blood andwas observed for a maximal 120 minute period.

Based on studies conducted using: a) inhibitor and NaCl; b) inhibitorand 0.1 mg/kg NASP; c) inhibitor and 0.4 mg/kg NASP; d) inhibitor and1.6 mg/kg NASP; and e) inhibitor and 300 U/kg FEIBA, the in vivo studiesby TEG analysis of whole blood from guinea pigs showed that NASP 0.4mg/kg and FEIBA 300 U/kg performed better (i.e., more procoagulantactivity) than NaCl.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the embodimentsshown and described herein. Rather, the scope and spirit of presentinvention is embodied by the appended claims.

1-73. (canceled)
 74. A method of identifying a tissue factor pathwayinhibitor (TFPI)-inhibiting compound which is capable of enhancing bloodcoagulation, the method comprising: contacting a first plasma samplecomprising TFPI with an antibody which inhibits TFPI activity; measuringpeak thrombin generation or peak time of the first plasma sample;contacting a second plasma sample comprising TFPI with a compositioncomprising a sulfated polysaccharide; measuring peak thrombin generationor peak time in the second plasma sample; and comparing the peakthrombin generation or peak time of the second plasma sample with thepeak thrombin generation or peak time of the first plasma sample,wherein no change in peak thrombin generation or peak time of the secondplasma sample compared to the first plasma sample indicates that thesulfated polysaccharide is a TFPI-inhibiting compound.
 75. The methodaccording to claim 74, wherein the antibody is an anti-TFPI C-terminusantibody.
 76. The method according to claim 74, wherein the first plasmasample and the second plasma sample are normal plasma.
 77. The methodaccording to claim 74, wherein the sulfated polysaccharide is afucoidan.
 78. The method according to claim 74, wherein the sulfatedpolysaccharide has a fucose content of 60% or greater.
 79. The methodaccording to claim 74, wherein the fucoidan is selected from the groupconsisting of Fucoidan 5307002, Fucus vesiculosus; Fucoidan L/FVF1091,Fucus vesiculosus; Fucoidan DS100159, Fucus vesiculosus; FucoidanDS100160, Fucus vesiculosus; and Fucoidan VG2010100, Fucus vesiculosus.80. The method according to claim 74, wherein the composition comprisingthe sulfated polysaccharide comprises an alginate content of 10% orless.
 81. The method according to claim 74, wherein the sulfatedpolysaccharide is Fucoidan L/FVF1091.
 82. A method of identifying aC-terminal TFPI-binding compound which is capable of enhancing bloodcoagulation, the method comprising: contacting a plasma samplecomprising full-length TFPI and an antibody directed against theC-terminus of TFPI with recombinant C-terminus truncated TFPI (TFPI1-160); determining a first peak thrombin generation or first peak timeof the plasma sample after contacting with recombinant C-terminustruncated TFPI (TFPI 1-160); contacting the plasma sample with acomposition comprising a sulfated polysaccharide; determining a secondpeak thrombin generation or second peak time after contacting the plasmasample with the composition comprising a sulfated polysaccharide; andcomparing the first peak thrombin generation or first peak time of theplasma sample with the second peak thrombin generation or second peaktime of the plasma sample, wherein an increase in peak thrombingeneration or decrease in peak time of the plasma sample indicates thatthe test compound is a C-terminal TFPI-binding compound.
 83. The methodaccording to claim 82, wherein the plasma sample is normal plasma. 84.The method according to claim 82, wherein the plasma sample isFVIII-inhibited plasma.
 85. The method according to claim 82, whereinthe sulfated polysaccharide is a fucoidan.
 86. The method according toclaim 82, wherein the sulfated polysaccharide has a fucose content of60% or greater.
 87. The method according to claim 82, wherein thefucoidan is selected from the group consisting of Fucoidan 5307002,Fucus vesiculosus; Fucoidan L/FVF1091, Fucus vesiculosus; FucoidanDS100159, Fucus vesiculosus; Fucoidan DS100160, Fucus vesiculosus; andFucoidan VG2010100, Fucus vesiculosus.
 88. The method according to claim82, wherein the composition comprising the sulfated polysaccharidecomprises an alginate content of 10% or less.
 89. The method accordingto claim 82, wherein the sulfated polysaccharide is Fucoidan L/FVF1091.90. A method of identifying a Factor XI-activation compound which iscapable of enhancing blood coagulation, the method comprising:contacting Factor XI and a fucoidan with a Factor-XI deficient plasma toproduce a first plasma sample; determining thrombin generation in thefirst plasma sample; contacting the first reaction mixture withFactor-XIa to produce a second plasma sample; determining the thrombingeneration in the second plasma sample; and comparing the thrombingeneration of the second plasma sample with the thrombin generation ofthe first plasma sample, wherein no change in the thrombin generation inthe second plasma sample compared to the first plasma sample indicatesthat the fucoidan is a Factor XI-activation compound.
 91. The methodaccording to claim 90, wherein Factor XI has a concentration of about 30nM.
 92. The method according to claim 90, wherein Factor XIa has aconcentration of about 60 pM.
 93. The method according to claim 90,wherein the fucoidan has a fucose content of 60% or greater.